Bioluminescent novelty items

ABSTRACT

Novelty items that are combinations of articles of manufacture with bioluminescence generating systems and/or fluorescent proteins are provided. These novelty items, which are articles of manufacture, are designed for entertainment, recreation and amusement, and include toys, personal items, such as cosmetics, bath powders, body lotions, gels, powders and creams, toothpastes and other dentifrices, soaps, body paints, and bubble bath, fountains, including liquid &#34;fireworks&#34; and other such jets or sprays or aerosols of compositions that are solutions, mixtures, suspensions, powders, pastes, particles or other formulations.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/135,988to Bruce Bryan, filed Aug. 17, 1998, entitled "BIOLUMINESCENT NOVELTYITEMS." This application is also continuation-in-part of U.S.application Ser. No. 08/757,046, now U.S. Pat. No. 5,876,995, to BruceBryan, filed Nov. 25, 1996, entitled "BIOLUMINESCENT NOVELTY ITEMS."This application is also a continuation-in-part of U.S. application Ser.No. 08/597,274, now allowed, to Bruce Bryan, filed Feb. 6, 1996,entitled "BIOLUMINESCENT NOVELTY ITEMS".

U.S. Pat. No. 09/135,988 is a continuation-in-part of U.S. applicationSer. No. 08/757,046, which is a continuation-in-part of U.S. applicationSer. No. 08/597,274. The subject matter of each of U.S. application Ser.Nos. 09/135,988, 08/597,274 and 08/757,046 is herein incorporated in itsentirety by reference thereto. This application is also related toprovisional application Ser. Nos. 60/079,624 and 60/089,367. Thedisclosures of each of the above noted applications and provisionalapplications is incorporated herein by reference thereto.

FIELD OF INVENTION

The present invention relates to systems for producing bioluminescentlight, and to combinations of the systems with articles of manufactureincluding toys, textiles, food and beverages, to produce novelty items.By virtue of the combination, the novelty items glow or produce or expela bioluminescent composition. Also, provided are compositions,encapsulated bioluminescence generating reagents, and methods forproducing the bioluminescence.

BACKGROUND OF THE INVENTION

Luminescence is a phenomenon in which energy is specifically channeledto a molecule to produce an excited state. Return to a lower energystate is accompanied by release of a photon (hγ). Luminescence includesfluorescence, phosphorescence, chemiluminescence and bioluminescence.Bioluminescence is the process by which living organisms emit light thatis visible to other organisms. Luminescence may be represented asfollows:

    A+B→X*+Y

    X*→X+hν,

where X* is an electronically excited molecule and hγ represents lightemission upon return of X* to a lower energy state. Where theluminescence is bioluminescence, creation of the excited state derivesfrom an enzyme catalyzed reaction. The color of the emitted light in abioluminescent (or chemiluminescent or other luminescent) reaction ischaracteristic of the excited molecule, and is independent from itssource of excitation and temperature.

An essential condition for bioluminescence is the use of molecularoxygen, either bound or free in the presence of a luciferase.Luciferases, are oxygenases, that act on a substrate, luciferin, in thepresence of molecular oxygen and transform the substrate to an excitedstate. Upon return to a lower energy level, energy is released in theform of light [for reviews see, e.g., McElroy et al. (1966) in MolecularArchitecture in Cell Physiology, Hayashi et al., eds., Prentice-Hall,Inc., Englewood Cliffs, N.J., pp. 63-80; Ward et al., Chapter 7 inChemi-and Bioluminescence, Burr, ed., Marcel Dekker, Inc. NY,pp.321-358; Hastings, J. W. in (1995) Cell Physiology: Source Book, N.Sperelakis (ed.), Academic Press, pp 665-681; Luminescence, Narcosis andLife in the Deep Sea, Johnson, Vantage Press, NY, see, esp. pp. 50-56].

Though rare overall, bioluminescence is more common in marine organismsthan in terrestrial organisms. Bioluminescence has developed from asmany as thirty evolutionarily distinct origins and, thus, is manifestedin a variety of ways so that the biochemical and physiologicalmechanisms responsible for bioluminescence in different organisms aredistinct. Bioluminescent species span many genera and includemicroscopic organisms, such as bacteria [primarily marine bacteriaincluding Vibrio species], fungi, algae and dinoflagellates, to marineorganisms, including arthropods, mollusks, echinoderms, and chordates,and terrestrial organism including annelid worms and insects.

Bioluminescence, as well as other types of chemiluminescence, is usedfor quantitative determinations of specific substances in biology andmedicine. For example, luciferase genes have been cloned and exploitedas reporter genes in numerous assays, for many purposes. Since thedifferent luciferase systems have different specific requirements, theymay be used to detect and quantify a variety of substances. The majorityof commercial bioluminescence applications are based on firefly[Photinus pyralis] luciferase. One of the first and still widely usedassays involves the use of firefly luciferase to detect the presence ofATP. It is also used to detect and quantify other substrates orco-factors in the reaction. Any reaction that produces or utilizesNAD(H), NADP(H) or long chain aldehyde, either directly or indirectly,can be coupled to the light-emitting reaction of bacterial luciferase.

Another luciferase system that has been used commercially for analyticalpurposes is the Aequorin system. The purified jellyfish photoprotein,aequorin, is used to detect and quantify intracellular Ca²⁺ and itschanges under various experimental conditions. The Aequorin photoproteinis relatively small [˜20 kDa], nontoxic, and can be injected into cellsin quantities adequate to detect calcium over a large concentrationrange [3×10⁻⁷ to 10⁻⁴ M].

Because of their analytical utility, many luciferases and substrateshave been studied and well-characterized and are commercially available[e.g., firefly luciferase is available from Sigma, St. Louis, Mo., andBoehringer Mannheim Biochemicals, Indianapolis, Ind.; recombinantlyproduced firefly luciferase and other reagents based on this gene or foruse with this protein are available from Promega Corporation, Madison,Wis.; the aequorin photoprotein luciferase from jellyfish and luciferasefrom Renilla are commercially available from Sealite Sciences, Bogart,Ga.; oelenterazine, the naturally-occurring substrate for theseluciferases, is available from Molecular Probes, Eugene, Oreg.]. Theseluciferases and related reagents are used as reagents for diagnostics,quality control, environmental testing and other such analyses. Thesereagents have not been used in connection with entertainment andrecreation for the glow, illumination and color produced upon generationof bioluminescence.

Thus, it is an object herein to exploit bioluminescence for use as arecreational product in combination with articles of manufacture toproduce novelty items, including toys, personal items, foods, fountains,beverages, coating compositions, such as paints and inks, textiles,including clothing, toy cigarettes, fish food, particularly for feedingtransgenic fish that express a luciferase, jewelry and other such items.It is also an object herein to provide such combinations and to providemeans for producing and using such combinations.

SUMMARY OF THE INVENTION

Systems and apparatus for generating bioluminescence, and combinationsof these systems and apparatus with inanimate articles of manufacture toproduce novelty items are provided. These novelty items, which arearticles of manufacture, are designed for entertainment, recreation andamusement, and include, but are not limited to: toys, particularlysquirt guns, toy cigarettes, toy "Halloween" eggs, footbags andboard/card games; finger paints and other paints, slimy play material;textiles, particularly clothing, such as shirts, hats and sports gearsuits, threads and yarns; bubbles in bubble making toys and other toysthat produce bubbles; balloons; figurines; personal items, such as bathpowders, body lotions, gels, powders and creams, nail polishes, make-up,toothpastes and other dentifrices, soaps, body paints, and bubble bath;items such as inks, paper; foods, such as gelatins, popcorn, icings andfrostings; fish food containing luciferins and transgenic fish,particularly transgenic fish that express a luciferase; plant foodcontaining a luciferin or luciferase, preferably a luciferin for usewith transgenic plants that express luciferase; and beverages, such asbeer, wine, champagne, soft drinks, and ice cubes and ice in otherconfigurations; fountains, including liquid "fireworks" and other suchjets or sprays or aerosols of compositions that are solutions, mixtures,suspensions, powders, pastes, particles or other suitable form.

Thus, the novelty items provided herein include but are not limited to:textiles that glow, ink that glows, paints, particularly fingerpaints,that glow, paper products that glow, toys, particularly reloadablesquirt guns that eject a bioluminescent fluid, dolls and dummies withinternal organs or parts that glow, figurines and novelty items thatglow; toy "cigarettes" that produce glowing "smoke" upon exhalation, toyeggs with glowing yolks and/or whites, toy footbags that glow and toyboard and card games with glowing parts, such as glowing cards, dice,game boards, etc.; foods and beverages that glow, soapy compositions forblowing bubbles that produce bubbles that glow, bubble bath compositionsthat produce bubbles that glow, fountains that expel glowing fluid,bioluminescent "fireworks", sparklers, magic-wand toys, and numerousother such items. Food containing a luciferin for use with plants andanimals that express luciferase, such as transgenic fish, then when feda food containing an appropriate substrate glow, is also contemplatedherein.

Bioluminescence is advantageously used in combination with such noveltyitems because it can be generated using reagents that are nontoxic,noncorrosive and nonstaining. Bioluminescence is also advantageouslyused because it can be sustained to provide a glow that lasts, ifdesired, from minutes up to hours.

Any article of manufacture that can be combined with abioluminescence-generating system as provided herein and thereby provideentertainment, recreation and/or amusement, including use of the itemsfor recreation or to attract attention, such as for advertising goodsand/or services that are associated with a logo or trademark iscontemplated herein. Such uses may be in addition to or in conjunctionwith or in place of the ordinary or normal use of such items. As aresult of the combination, the items glow or produce, such as in thecase of squirt guns and fountains, a glowing fluid or spray of liquid orparticles. The novelty in the novelty item derives from itsbioluminescence.

The preferred bioluminescence-generating reactions are performed byadding oxygen (or water containing oxygen) or calcium ions or otherappropriate metal ion to luciferin and luciferase mixtures usingapparatus and systems as described herein. Apparatus, systems andsubstrates for generating the bioluminescence are provided. The systemsinclude matrix materials that are coated with bioluminescence generatingreagents, capsular vehicles containing the reagents and single chamberand multiple chamber apparatus containing the reagents. The matrixmaterials are used, for example, in the fabrication of clothing itemsand also in the loading cartridges described herein.

Methods and compositions for producing bioluminescence in combinationwith the novelty items are also provided. Micro- and macro-capsularvehicles containing bioluminescence generating reagents are provided.The capsular vehicles are capsules, such as liposomes, isolatedendosomes, isolated vacuoles, gelatin capsules, and other such deliveryvehicles, and the apparatus include vessels, and single chamber, dualchamber and three chamber or more apparatus. These vehicles encapsulatebioluminescence generating system reagents, and typically contain lessthan all of the reagents necessary to generate a bioluminescentreaction. The capsular vehicles include vehicles often used for drugdelivery, such as liposomes, and time release capsules; and alsocapsules made of glass, plastic and other such materials.

For example, the bioluminescence generating reagents (or components) maybe coated on the inside of a glass container, such as a glass capillarytube [see, e.g., U.S. Pat. No. 5,387,526]. Upon addition of acomposition containing the necessary activating agents, such asmolecular oxygen, ATP, a reductase, Ca²⁺ [or other suitable metal ion],the coating will be contacted with the activator and will produce aglow. The capsular vehicles are intended for use in combination with thearticles of manufacture.

Thus, the micro- or macro-capsular vehicles, when crushed, opened,dissolved or otherwise placed under conditions that cause delivery ofthe contents, release material that glows upon contact with air and/ormoisture and/or other activator(s). These vehicles vary in size [in thelargest dimension] from as small as less than 0.1 μm up to 0.1 cm ormore.

Matrix materials, such as glass, plastics, cotton and other textilematerial, that contain linked bioluminescence-generating reagents arealso provided. For example, one or more components of thebioluminescence generating system is (are) linked by adsorption,absorption or other means, directly or indirectly (such as via a linker)to a matrix material. Matrix materials, such as textiles, glass, plasticor ceramic surfaces or particles adapted for linking molecules, forexample such as luciferases or luciferins, are combined with at leastone component of the bioluminescence generating system, particularly theluciferin, luciferase, or, where the components are amenable, theluciferin and luciferase. The component(s) such as the luciferase arelinked to the matrix, such as cotton, using methods known to those ofskill in the art of protein synthesis for linking peptides or proteinsto solid substrates [see, e.g., Eichler et al. (1993) Biochemistry32:11035-11041; Merrifield (1964) Biochemistry 3:1385-1390.] Linkage iseffected either covalently or non-covalently and can be direct or vialinkers. Such methods and linkers are well known to those of skill inthe chemical arts. The matrix materials with linked bioluminescencegenerating system components are contacted with an article ofmanufacture resulting in a novelty item that, when appropriatelytreated, such as by spraying on a composition that contains theremaining components of the reactions, glows or producesbioluminescence. The matrix materials are advantageously used in theloading cartridges provided herein.

Also provided are single and multi-chamber, particularly dual chamber,apparatus for producing bioluminescence, and combinations of theseapparatus with bioluminescence generating reagents are also provided.Such apparatus include at least one chamber that contains all but atleast one reagent or component required to produce bioluminescence. Uponaddition of the component either to the chamber or after ejection ofsome or all of the contents of the chamber a bioluminescent glow orglowing fluid, spray or jet is produced. Recharging or chargingcartridges adapted for loading these apparatus are also provided.

The charging, or recharging, cartridges are designed to be used to loadcomponents of a bioluminescence generating system into or onto anarticle of manufacture to produce the novelty items, and also to permitreuse after the bioluminescence generating system is spent. Thecartridge, which contains one or more chambers, is in an exemplaryembodiment fabricated with two-chambers. In a preferred embodiment, thecartridge includes a matrix material, such as a porous membrane or acotton ball to which a bioluminescence generating agent, such as aluciferase or luciferin, is adsorbed or absorbed such that when flushedwith an appropriate composition will be released from the matrix. Thefirst chamber contains one or more components of a bioluminescencegenerating system used in the bioluminescent process, and the secondchamber contains a composition that will flush or otherwise desorb aquantity of the component from the matrix material. Typically, thecomposition is contained in an easily puncturable or compressible vialand positioned adjacent to the matrix material. In operation, a plunger,a dual pronged plunger where there are two or more chambers, is alignedso that one prong of the plunger is positioned in each chamber, or theplunger may be movably attached to the cartridge, and the output nozzlesof the cartridge are aligned against the filler ports of a novelty item,such as a squirt gun. The plunger is then forced into the cartridge,thereby dispensing the components out the nozzle of the first chamberand into the first chamber in the novelty item, and compressing the vialof fluid to flush the remaining components of the bioluminescencegenerating system from the nozzle of the second chamber and into thesecond chamber of the novelty item. In this manner, the novelty itemscontemplated herein may be initially charged, or recharged again andagain, by replenishing any or all of the components necessary forgenerating bioluminescence.

Articles of manufacture containing one or more components of abioluminescence generating system or a composition, such as acomposition containing ATP or Ca²⁺ or other activator, within thepackaging material, and a label that indicates that the contents is usedfor generating bioluminescence are also provided.

Kits containing an article of manufacture and appropriate reagents forgenerating bioluminescence are also provided.

DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 is a side elevation, with portions cut away, of a squirt gunincorporating the dual chamber structure;

FIG. 2 is a sectional view taken on line 2--2 of FIG. 1;

FIG. 3 is a sectional view taken on line 3--3 of FIG. 1;

FIG. 4 is a side elevation view, with portions cut away, of a gaspowered toy gun with dual chamber detachable fluid reservoir;

FIG. 5 is a top plan view of the toy gun of FIG. 4, with portions cutaway;

FIG. 6 is a side elevation view, partially cut away of a gas-chargedfluid dispensing apparatus incorporating the dual chamber system;

FIG. 7 is a sectional view taken on line 7--7 of FIG. 6;

FIG. 8 is a top plan view of the structure of FIG. 6, partially cutaway;

FIG. 9 is a side elevation view of a fountain type configuration of thegas-charged dual chamber fluid dispensing apparatus, with portions cutaway;

FIG. 10 is a sectional view taken on line 10--10 of FIG. 9;

FIG. 11 is a side elevation view, partially cut away, of a dual chambercompressible dispensing container;

FIG. 12 is a side elevation view, partially cut away of a bottle/bladderapparatus designed for use with bubble-blowing compositions;

FIG. 13 is a view similar to FIG. 12, with the components mixed and thebubble blowing wand detached for use; and

FIG. 14 is a side elevation view, partially cut away, of beveragecontainer with a bladder apparatus actuated by opening of the beveragecontainer.

FIG. 15 is a side elevation view, partially cut away of a single use,dual chamber fluid packaging apparatus adapted for use withbubble-blowing compositions.

FIG. 16 is a side elevation view, partially cut away of a cap apparatusoperated by depression of the plunger assembly to rupture the capsulecontained within the cork cap.

FIG. 17 is a side elevation view, partially cut away of a cap apparatusoperated by screwing the plunger assembly into the cork cap to rupturethe capsule contained therein.

FIG. 18 is a side elevation view, partially cut away of a cap apparatusoperated by screwing the screw-cap onto the top of the bottle forcingthe plunger assembly against the capsule contained within the neck ofthe bottle, thereby rupturing the capsule membranes.

FIG. 19 is a view similar to the view of FIG. 18, with the cap apparatustightly secured against the top of the bottle and the capsule membranesruptured.

FIG. 20 is a side elevation view, with portions cut away, of a spraycontainer or can in which the bottom portion of the apparatus is notengaged.

FIG. 21 is a side elevation view, with portions cut away, of the spraycontainer in which the bottom portion of the container is engaged.

FIG. 22 is a side elevation view of an exemplary pellet that containsbioluminescence-generating reagents and that is adapted for use with thespray container.

FIG. 23 is a side elevation, with portions cut away, of anotherembodiment of a squirt gun incorporating the dual chamber structure;

FIG. 24 is a top view, with portions cut away, of the nozzle end of thesquirt gun of FIG. 23;

FIG. 25 is a sectional view taken on line 25--25 of FIG. 23; and

FIG. 26 is a sectional view taken on line 26--26 of FIG. 23.

FIG. 27 is a side elevation view of a compressible tube configurationwith a portion cut away.

FIG. 28 is a pictorial view of a charging, or recharging, cartridge;

FIG. 29 is a sectional view taken on line 29--29 of FIG. 28, with theplunger in the starting position;

FIG. 30 is a sectional view similar to FIG. 29, showing the cartridgecontents ejected into receiving chambers of a typical unit as shown inFIG. 2;

FIG. 31 is a sectional view similar to FIG. 29, showing a plungerlocking device;

FIG. 32 is a sectional view similar to FIG. 30, showing the lockingdevice released to allow compression of the plunger;

FIG. 33 is a sectional view taken along line 33--33 of FIG. 31 andshowing the positioning of the locking device; and

FIG. 34 is a sectional view of an alternative embodiment dual chamberrefill cartridge.

DETAILED DESCRIPTION OF THE INVENTION Table of Contents

A. Definitions

B. Bioluminescence generating systems

1. General description

a. Luciferases

b. Luciferins

c. Activators

d. Reactions

2. Ctenophore and coelenterate systems

a. The aequorin system

(1) Aequorin photoprotein

(2) Luciferin

b. The Renilla system

3. Crustacean, particular Cyrpidina [Vargula], systems

a. Vargula luciferase

(1) Purification from Cypridina

(2) Preparation by Recombinant Methods

b. Vargula luciferin

c. Reaction

4. Insect bioluminescence generating systems including fireflies, clickbeetles, and other insect systems

a. Luciferase

b. Luciferin

c. Reaction

5. Bacterial systems

a. Luciferases

b. Luciferins

c. Reactions

6. Other systems

a. Dinoflagellate bioluminescence generating systems

b. Systems from molluscs, such as Latia and Pholas

c. Earthworms and other annelids

d. Glow worms

e. Marine polycheate worm systems

f. South American railway beetle

g. Fish

7. Fluorescent proteins

a. Green and blue fluorescent proteins

b. Phycobiliproteins

C. Practice of the reactions in combination with articles of manufacture

D. Packaging of Bioluminescence Systems

1. Dispensing and Packaging Apparatus for Combination with theBioluminescence Generating System Components

2. Capsules, pellets, liposomes, micronized particles

a. Encapsulating vehicles--in general

b. Encapsulating vehicles--liposomes

c. Encapsulating vehicles--gelatin and polymeric vehicles

d. Endosomes and vacuoles

e. Micronized particles

3. Apparatus and substrates

a. Matrix materials

b. Immobilization and activation

4. Apparatus containing a single chamber, housing or a vessel

5. Dual and multiple chamber fluid dispensing apparatus

a. Mechanical pump dispensing apparatus

b. Gas-charged dispensing apparatus

c. Compressible dispensing apparatus

6. Other fluid dispensing and packaging apparatus particularly designedfor single or multiple uses

a. Bottle-type single chamber container/bladder apparatus

b. Dual chambered bottle type container/bladder apparatus for use withfoods and beverages

c. Can type container/bladder apparatus for use with foods and beverages

d. Spray containers for use to produce a glowing spray

7. Cap Apparatus for use a single chamber vessel

E. Combinations of articles of manufacture and bioluminescence

1. Personal care products, including bath powders, bubble baths,products for use on the nails, hair, skin, lips and elsewhere

a. Bath powders

b. Glowing dust or powder

c. Lotions, gels and other topical application formulations

(1) Lotions

(2) Creams

(3) Solutions and suspensions for topical application

(4) Gels

(5) Solids

2. Glowing toys and other items

a. Single chamber toy guns and other toy weapons that shoot pellets orliquid

b. Bubble-making toys

c. Board/Card games

d. Toy Eggs

e. Footbags, bean bags and balls

f. Figurines

3. Glowing textiles and paper products

4. Foods and beverages, including ice cubes

a. Beverages

b. Ice

c. Popcorn

5. Jewelry, Clothing and Other Items of Manufacture

6. Fountains

7. Non-Tobacco Toy Cigarettes

8. Fish, Fish Bait and Fish Food

9. Plant Food and Animal Food

F. Cartridges for loading (charging or filling) or reloading(recharging) the novelty items

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publications ofreferred to herein are incorporated by reference in their entirety.

As used herein, novelty items refer to inanimate articles of manufacturethat are intended to provide, even for only a few moments, amusement,entertainment, decoration or recreation. The use for recreation orentertainment may be the items only use or may be in addition to otheruses or benefits of the items, such as clothing that is modified, asdescribed herein, by combination with bioluminescence.

Novelty items are understood by those of skill in manufacture of suchitems as well as by the purchasing public and are intended herein toinclude items, such as, toys, including toy guns, dolls, dummies,figurines, balloons, bubbles, "fairy dust", such as micronizedlyophilized particles, puzzles, and inks and paints, particularlyfingerpaints; theatrical vapors when mixed, for example with dry ice ora fog; souvenirs; textiles, particularly clothing, including T-shirts,hats, swimsuits, bathing suit, wet suits, scuba diving suits, surfingsuits, and other water sport or sports attire; foods and beverages,including gelatins, ice cubes and ice in other shapes, beer, wine,champagne, soft drinks, ice creams, sorbets, ices, frostings, and candy;jewelry, medallions, decorative articles, artificial flowers, articlesfor displaying names, business trade names, slogans, trademarks onpromotional or other such items, such as T-shirts, hats, paints,wrapping paper, gifts intended to promote business goodwill; personalitems, such as body paints, body sprays, bubble baths, make-up, bodylotions, dentifrices; fountains; jets or sprays of particles or fluids,including "fireworks", sparklers, and magic-wand toys, and many othersuch novelty items [see, e.g., U.S. Pat. Nos. 5,435,010, 5,460,022,5,458,931, 5,435,787, 5,435,010, 5,432,623, 5,421,583, 5,419,558,5,416,927, 5,413,454, 5,413,332, 5,411,427, 5,410,962, 5,407,691,5,407,391, 5,405,958, 5,405,206, 5,400,698, 5,399,122, 5,398,972,5,397,609, 5,396,408, 5,393,580, 5,390,086, 5,389,033, 5,383,684,5,374,805, 5,368,518, 5,363,984, 5,360,010, 5,353,378, 5,351,931,5,346,455, 5,341,538, 5,323,492, 5,283,911, 5,222,797, 5,177,812,5,158,349, 4,924,358, 3,597,877 and many others, which describe types ofitems are considered novelty items]. Any such inanimate item that iscombined with bioluminescence is intended to be encompassed herein.

Thus, for purposes herein, a novelty item refers to any inanimatearticle of manufacture that, upon combination with bioluminescence,provides amusement, entertainment, recreation or enjoyment, if only foreven a few moments. Addition of the bioluminescence to the article ofmanufacture does not add to the function of the item, but addsentertainment, amusement or recreational aspects to the item so that theresulting combination is a novelty item. Therefore, the combinationsprovided herein are novelty items by virtue of the combination of aninanimate article of manufacture with bioluminescence.

As used herein, inanimate means that the articles of manufacture are notalive nor formerly living [i.e., dead] items. Thus, the novelty itemsherein, do not encompass living organisms, such as genetically modifiedfireflies or genetically engineered plants that express luciferase orother such organisms that produce bioluminescence. Animal food and plantfood containing luciferin (or luciferase) and/or other activators foruse with a transgenic animal or plant that expresses the correspondingluciferase (or luciferin) are provided. These are intended to result inan illuminated animal or plant upon ingestion or consumption orabsorption of the food. Transgenic fish and food therefor are alsoprovided herein.

As used herein, personal items include items that are used on the body,such as toothpastes, dentifrices, make-up, nail polishes, body lotions,body creams, body paints and body powders.

As used herein, chemiluminescence refers to a chemical reaction in whichenergy is specifically channeled to a molecule causing it to becomeelectronically excited and subsequently to release a photon therebyemitting visible light. Temperature does not contribute to thischanneled energy. Thus, chemiluminescence involves the direct conversionof chemical energy to light energy. Bioluminescence refers to the subsetof chemiluminescence reactions that involve luciferins and luciferases(or the photoproteins). Bioluminescence does not herein includephosphorescence.

As used herein, "fairy dust" refers to particles, such as lightsensitive liposomes or micronized powdered particles, that glow uponcontact with the air, such as "dust" that a child would use whenpretending to be Tinker Bell or other such character.

As used herein, reference to ice cubes include ice in any shape or form,including, but not limited to: cubes; ice formations made from precastmolds, such as figurines, icicles, ice sculptures and other such noveltyitems formed from ice.

As used herein, luminescence refers to the detectable EM radiation,generally, UV, IR or visible EM radiation that is produced when theexcited product of an exergic chemical process reverts to its groundstate with the emission of light. Chemiluminescence is luminescence thatresults from a chemical reaction. Bioluminescence is chemiluminescencethat results from a chemical reaction using biological molecules [orsynthetic versions or analogs thereof] as substrates and/or enzymes.

As used herein, bioluminescence, which is a type of chemiluminescence,refers to the emission of light by biological molecules, particularlyproteins. The essential condition for bioluminescence is molecularoxygen, either bound or free in the presence of an oxygenase, aluciferase, which acts on a substrate, a luciferin. Bioluminescence isgenerated by an enzyme or other protein [luciferase] that is anoxygenase that acts on a substrate luciferin [a bioluminescencesubstrate] in the presence of molecular oxygen and transforms thesubstrate to an excited state, which upon return to a lower energy levelreleases the energy in the form of light.

As used herein, the substrates and enzymes for producing bioluminescenceare generically referred to as luciferin and luciferase, respectively.When reference is made to a particular species thereof, for clarity,each generic term is used with the name of the organism from which itderives, for example, bacterial luciferin or firefly luciferase.

As used herein, luciferase refers to oxygenases that catalyze a lightemitting reaction. For instance, bacterial luciferases catalyze theoxidation of flavin mononucleotide [FMN] and aliphatic aldehydes, whichreaction produces light. Another class of luciferases, found amongmarine arthropods, catalyzes the oxidation of Cypridina [Vargula]luciferin, and another class of luciferases catalyzes the oxidation ofColeoptera luciferin.

Thus, luciferase refers to an enzyme or photoprotein that catalyzes abioluminescent reaction [a reaction that produces bioluminescence]. Theluciferases, such as firefly and Renilla luciferases, that are enzymeswhich act catalytically and are unchanged during the bioluminescencegenerating reaction. The luciferase photoproteins, such as the aequorinand obelin photoproteins to which luciferin is non-covalently bound, arechanged, such as by release of the luciferin, during bioluminescencegenerating reaction. The luciferase is a protein that occurs naturallyin an organism or a variant or mutant thereof, such as a variantproduced by mutagenesis that has one or more properties, such as thermalor pH stability, that differ from the naturally-occurring protein.Luciferases and modified mutant or variant forms thereof are well known.

Thus, reference, for example, to "Renilla luciferase" means an enzymeisolated from member of the genus Renilla or an equivalent moleculeobtained from any other source, such as from another Anthozoa, or thathas been prepared synthetically.

The luciferases and luciferin and activators thereof are referred to asbioluminescence generating reagents or components. Typically, a subsetof these reagents will be provided or combined with an article ofmanufacture. Bioluminescence will be produced upon contacting thecombination with the remaining reagents. Thus, as used herein, thecomponent luciferases, luciferins, and other factors, such as O₂, Mg²⁺,Ca²⁺ are also referred to as bioluminescence generating reagents [oragents or components].

As used herein, "not strictly catalytically" means that the photoproteinacts as a catalyst to promote the oxidation of the substrate, but it ischanged in the reaction, since the bound substrate is oxidized and boundmolecular oxygen is used in the reaction. Such photoproteins areregenerated by addition of the substrate and molecular oxygen underappropriate conditions known to those of skill in this art.

As used herein, bioluminescence substrate refers to the compound that isoxidized in the presence of a luciferase, and any necessary activators,and generates light. These substrates are referred to as luciferins,which are substrates that undergo oxidation in a bioluminescencereaction. These bioluminescence substrates include any luciferin oranalog thereof or any synthetic compound with which a luciferaseinteracts to generate light. Preferred substrates are those that areoxidized in the presence of a luciferase or protein in alight-generating reaction. Bioluminescence substrates, thus, includethose compounds that those of skill in the art recognize as luciferins.Luciferins, for example, include firefly luciferin, Cypridina [alsoknown as Vargula] luciferin [coelenterazine], bacterial luciferin, aswell as synthetic analogs of these substrates or other compounds thatare oxidized in the presence of a luciferase in a reaction the producesbioluminescence.

As used herein, capable of conversion into a bioluminescence substratemeans susceptible to chemical reaction, such as oxidation or reduction,that yields a bioluminescence substrate. For example, the luminescenceproducing reaction of bioluminescent bacteria involves the reduction ofa flavin mononucleotide group (FMN) to reduced flavin mononucleotide(FMNH₂) by a flavin reductase enzyme. The reduced flavin mononucleotide[substrate] then reacts with oxygen [an activator] and bacterialluciferase to form an intermediate peroxy flavin that undergoes furtherreaction, in the presence of a long-chain aldehyde, to generate light.With respect to this reaction, the reduced flavin and the long chainaldehyde are substrates.

As used herein, bioluminescence system [or bioluminescence generatingsystem] refers to the set of reagents required for abioluminescence-producing reaction. Thus, the particular luciferase,luciferin and other substrates, solvents and other reagents that may berequired to complete a bioluminescent reaction form a bioluminescencesystem. Therefore, a bioluminescence system (or equivalently abioluminescence generating system) refers to any set of reagents that,under appropriate reaction conditions, yield bioluminescence.Appropriate reaction conditions refers to the conditions necessary for abioluminescence reaction to occur, such as pH, salt concentrations andtemperature. In general, bioluminescence systems include abioluminescence substrate (a luciferin), a luciferase, which includesenzymes luciferases and photoproteins, and one or more activators. Aparticular bioluminescence system may be identified by reference to thespecific organism from which the luciferase derives; for example, theVargula [also called Cypridina] bioluminescence system (or Vargulasystem) includes a Vargula luciferase, such as a luciferase isolatedfrom the ostracod, Vargula or produced using recombinant means ormodifications of these luciferases. This system would also include theparticular activators necessary to complete the bioluminescencereaction, such as oxygen and a substrate with which the luciferasereacts in the presence of the oxygen to produce light.

As used herein, recharging or reloading the item refers to the means bywhich spent bioluminescence generating components are added to an item.Recharging generally refers to a process in which one component, such asa luciferase is added to an item, such as a textile; reloading refers tothe process in which all components are added to an item, such as arefillable squirt gun.

As used herein, ATP, AMP, NAD+ and NADH refer to adenosine triphosphate,adenosine monophosphate, nicotinamide adenine dinucleotide (oxidizedform) and nicotinamide adenine dinucleotide (reduced form),respectively.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

As used herein equivalent, when referring to two sequences of nucleicacids means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When "equivalent" is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with onlyconservative amino acid substitutions [see, e.g., Table 2, below] thatdo not substantially alter the activity or function of the protein orpeptide. When "equivalent" refers to a property, the property does notneed to be present to the same extent [e.g., two peptides can exhibitdifferent rates of the same type of enzymatic activity], but theactivities are preferably substantially the same. "Complementary," whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, preferably with less than 25%,more preferably with less than 15%, even more preferably with less than5%, most preferably with no mismatches between opposed nucleotides.Preferably the two molecules will hybridize under conditions of highstringency.

As used herein: stringency of hybridization in determining percentagemismatch is as follows:

1) high stringency: 0.1× SSPE, 0.1% SDS, 65° C.

2) medium stringency: 0.2× SSPE, 0.1% SDS, 50° C.

3) low stringency: 1.0× SSPE, 0.1% SDS, 50° C.

It is understood that equivalent stringencies may be achieved usingalternative buffers, salts and temperatures.

The term "substantially" varies with the context as understood by thoseskilled in the relevant art and generally means at least 70%, preferablymeans at least 80%, more preferably at least 90%, and most preferably atleast 95%.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon administration ofa compound, composition or other mixture. Biological activities may beobserved in in vitro systems designed to test or use such activities.Thus, for purposes herein the biological activity of a luciferase is itsoxygenase activity whereby, upon oxidation of a substrate, light isproduced.

As used herein, a composition refers to a any mixture. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a combination refers to any association between two oramong more items.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, plant food refers to any liquids, water-soluble orwater-insoluble solids, such as fertilizers containing any ratio ofnitrogen, potassium and/or phosphorous, formulations, combinations,polymers or plant growth promoters, such as auxins and hormones, that isapplied to a plant to promote or maintain growth [e.g., see U.S. Pat.Nos. 4,016,880, 4,711,659, 4,804,403, 5,547,486, 5,553,853, RE 35,320,and RE 31,801]. The plant food may be applied directly to the soil,sprayed on the foliage of the plant or a combination thereof. The plantfood may be slow releasing or available immediately for consumption bythe plant. The plant food may be applied to any plant that can begenetically engineered to contain a heterologous gene encoding acomponent of a bioluminescence generating system, preferably aluciferase. Examples of such plants, but not meant to be limiting to,are grasses, agricultural plants and ornamental plants.

B. Bioluminescence Generating Systems

A bioluminescence generating system refers to the components that arenecessary and sufficient to generate bioluminescence. These include aluciferase, luciferin and any necessary co-factors or conditions.Virtually any bioluminescence generating system known to those of skillin the art will be amenable to use in the apparatus, systems,combinations and methods provided herein. Factors for consideration inselecting a bioluminescence generating system, include, but are notlimited to: the item used in combination with the bioluminescence; themedium in which the reaction is run; stability of the components, suchas temperature or pH sensitivity; shelf life of the components;sustainability of the light emission, whether constant or intermittent;availability of components; desired light intensity; and other suchfactors.

1. General Description

In general, bioluminescence refers to an energy-yielding chemicalreaction in which a specific chemical substrate, a luciferin, undergoesoxidation, catalyzed by an enzyme, a luciferase. Bioluminescentreactions are easily maintained, requiring only replenishment ofexhausted luciferin or other substrate or cofactor or other protein, inorder to continue or revive the reaction. Bioluminescence generatingreactions are well known to those of skill in this art and any suchreaction may be adapted for use in combination with articles ofmanufacture as described herein.

There are numerous organisms and sources of bioluminescence generatingsystems, and some representative genera and species that exhibitbioluminescence are set forth in the following table [reproduced in partfrom Hastings in (1995) Cell Physiology: Source Book, N. Sperelakis(ed.), Academic Press, pp 665-681]:

                  TABLE 1                                                         ______________________________________                                        Representative luminous organism                                              Type of Organism    Representative genera                                     ______________________________________                                        Bacteria            Photobacterium                                                                Vibrio                                                                        Xenorhabdus                                               Mushrooms           Panus, Armillaria                                                             Pleurotus                                                 Dinoflagellates     Gonyaulax                                                                     Pyrocystis                                                                    Noctiluca                                                 Cnidaria (coelenterates)                                                      Jellyfish           Aequorea                                                  Hydroid             Obelia                                                    Sea Pansy           Renilla                                                   Ctenophores         Mnemiopsis                                                                    Beroe                                                     Annelids                                                                      Earthworms          Diplocardia                                               Marine polychaetes  Chaetopterus, Phyxotrix                                   Syllid fireworm     Odontosyllis                                              Molluscs                                                                      Limpet              Latia                                                     Clam                Pholas                                                    Squid               Heteroteuthis                                                                 Heterocarpus                                              Crustacea                                                                     Ostracod            Vargula (Cypridina)                                       Shrimp (euphausids) Meganyctiphanes                                                               Acanthophyra                                                                  Oplophorus                                                                    Gnathophausia                                             Decapod             Sergestes                                                 Copepods                                                                      Insects                                                                       Coleopterids (beetles)                                                        Firefly             Photinus, Photuris                                        Click beetles       Pyrophorus                                                Railroad worm       Phengodes, Phrixothrix                                    Diptera (flies)     Arachnocampa                                              Echinoderms                                                                   Brittle stars       Ophiopsila                                                Sea cucumbers       Laetmogone                                                Chordates                                                                     Tunicates           Pyrosoma                                                  Fish                                                                          Cartilaginous       Squalus                                                   Bony                                                                          Ponyfish            Leiognathus                                               Flashlight fish     Photoblepharon                                            Angler fish         Cryptopsaras                                              Midshipman          Porichthys                                                Lantern fish        Benia                                                     Shiny loosejaw      Aristostomias                                             Hatchet fish        Agyropelecus                                              and other fish      Pachystomias                                                                  Malacosteus                                               Midwater fish       Cyclothone                                                                    Neoscopelus                                                                   Tarletonbeania                                            ______________________________________                                    

Other bioluminescent organisms contemplated for use herein areGonadostomias, Gaussia, Halisturia, Vampire squid, Glyphus, Mycotophids(fish), Vinciguerria, Howella, Florenciella, Chaudiodus, Melanocostusand Sea Pens.

It is understood that a bioluminescence generating system may beisolated from natural sources, such as those in the above Table, or maybe produced synthetically. In addition, for uses herein, the componentsneed only be sufficiently pure so that mixture thereof, underappropriate reaction conditions, produces a glow. Thus it has beenfound, in some embodiments, a crude extract or merely grinding up theorganism may be adequate. Generally, however, substantially purecomponents are used, but, where necessary, the precise purity can bedetermined empirically. Also, components may be synthetic componentsthat are not isolated from natural sources. DNA encoding luciferases isavailable [see, e.g., SEQ ID Nos. 1-13] and has been modified [see,e.g., SEQ ID Nos. 3 and 10-13] and synthetic and alternative substrateshave been devised. The DNA listed herein is only representative of theDNA encoding luciferases that is available.

Any bioluminescence generating system, whether synthetic or isolatedform natural sources, such as those set forth in Table 1, elsewhereherein or known to those of skill in the art, is intended for use in thecombinations, systems and methods provided herein. Chemiluminescencesystems per se, which do not rely on oxygenases [luciferases] are notencompassed herein.

a. Luciferases

Luciferases refer to any compound that, in the presence of any necessaryactivators, catalyze the oxidation of a bioluminescence substrate[luciferin] in the presence of molecular oxygen, whether free or bound,from a lower energy state to a higher energy state such that thesubstrate, upon return to the lower energy state, emits light. Forpurposes herein, luciferase is broadly used to encompass enzymes thatact catalytically to generate light by oxidation of a substrate and alsophotoproteins, such as aequorin, that act, though not strictlycatalytically [since such proteins are exhausted in the reaction], inconjunction with a substrate in the presence of oxygen to generatelight. These luciferases, including photoproteins, such as aequorin, areherein also included among the luciferases. These reagents include thenaturally-occurring luciferases [including photoproteins], proteinsproduced by recombinant DNA, and mutated or modified variants thereofthat retain the ability to generate light in the presence of anappropriate substrate, co-factors and activators or any other suchprotein that acts as a catalyst to oxidize a substrate, whereby light isproduced.

Generically, the protein that catalyzes or initiates the bioluminescentreaction is referred to as a luciferase, and the oxidizable substrate isreferred to as a luciferin. The oxidized reaction product is termedoxyluciferin, and certain luciferin precursors are termed etioluciferin.Thus, for purposes herein bioluminescence encompasses light produced byreactions that are catalyzed by [in the case of luciferases that actenzymatically] or initiated by [in the case of the photoproteins, suchas aequorin, that are not regenerated in the reaction] a biologicalprotein or analog, derivative or mutant thereof.

For clarity herein, these catalytic proteins are referred to asluciferases and include enzymes such as the luciferases that catalyzethe oxidation of luciferin, emitting light and releasing oxyluciferin.Also included among luciferases are photoproteins, which catalyze theoxidation of luciferin to emit light but are changed in the reaction andmust be reconstituted to be used again. The luciferases may be naturallyoccurring or may be modified, such as by genetic engineering to improveor alter certain properties. As long as the resulting molecule retainsthe ability to catalyze the bioluminescent reaction, it is encompassedherein.

Any protein that has luciferase activity [a protein that catalyzesoxidation of a substrate in the presence of molecular oxygen to producelight as defined herein] may be used herein. The preferred luciferasesare those that are described herein or that have minor sequencevariations. Such minor sequence variations include, but are not limitedto, minor allelic or species variations and insertions or deletions ofresidues, particularly cysteine residues. Suitable conservativesubstitutions of amino acids are known to those of skill in this art andmay be made generally without altering the biological activity of theresulting molecule. Those of skill in this art recognize that, ingeneral, single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBenjamin/Cummings Pub. co., p.224). Such substitutions are preferablymade in accordance with those set forth in TABLE 2 as follows:

                  TABLE 2                                                         ______________________________________                                        Original residue  Conservative substitution                                   ______________________________________                                        Ala (A)           Gly; Ser                                                    Arg (R)           Lys                                                         Asn (N)           Gln; His                                                    Cys (C)           Ser; neutral amino acid                                     Gln (Q)           Asn                                                         Glu (E)           Asp                                                         Gly (G)           Ala; Pro                                                    His (H)           Asn; Gln                                                    Ile (I)           Leu; Val                                                    Leu (L)           Ile; Val                                                    Lys (K)           Arg; Gln; Glu                                               Met (M)           Leu; Tyr; Ile                                               Phe (F)           Met; Leu; Tyr                                               Ser (S)           Thr                                                         Thr (T)           Ser                                                         Trp (W)           Tyr                                                         Tyr (Y)           Trp; Phe                                                    Val (V)           Ile; Leu                                                    ______________________________________                                    

Other substitutions are also permissible and may be determinedempirically or in accord with known conservative substitutions. Any suchmodification of the polypeptide may be effected by any means known tothose of skill in this art.

The luciferases may be obtained commercially, isolated from naturalsources, expressed in host cells using DNA encoding the luciferase, orobtained in any manner known to those of skill in the art. For purposesherein, crude extracts obtained by grinding up selected source organismsmay suffice. Since large quantities of the luciferase may be desired,isolation of the luciferase from host cells is preferred. DNA for suchpurposes is widely available as are modified forms thereof.

Examples of luciferases include, but are not limited to, those isolatedfrom the ctenophores Mnemiopsis (mnemiopsin) and Beroe ovata (berovin),those isolated from the coelenterates Aequorea (aequorin), Obelia(obelin), Pelagia, the Renilla luciferase, the luciferases isolated fromthe mollusca Pholas (pholasin), the luciferases isolated from theAristostomias and Porichthys fish and from the ostracods, such asCypridina (also referred to as Vargula). Preferred luciferases for useherein are the Aequorin protein, Renilla luciferase and Cypridina [alsocalled Vargula] luciferase [see, e.g., SEQ ID Nos. 1, 2, and 4-13].Also, preferred are luciferases which react to produce red and/or nearinfrared light. These include luciferases found in species ofAristostomias, such as A. scintillans, Pachystomias, Malacosteus, suchas M. niger.

b. Luciferins

The substrates for the reaction include any molecule(s) with which theluciferase reacts to produce light. Such molecules include thenaturally-occurring substrates, modified forms thereof, and syntheticsubstrates [see, e.g., U.S. Pat. Nos. 5,374,534 and 5,098,828].Exemplary luciferins include those described herein, as well asderivatives thereof, analogs thereof, synthetic substrates, such asdioxetanes [see, e.g., U.S. Pat. Nos. 5,004,565 and 5,455,357], andother compounds that are oxidized by a luciferase in a light-producingreaction [see, e.g., U.S. Pat. Nos. 5,374,534, 5,098,828 and 4,950,588].Such substrates also may be identified empirically by selectingcompounds that are oxidized in bioluminescent reactions.

c. Activators

The bioluminescence generating systems also require additionalcomponents discussed herein and known to those of skill in the art. Allbioluminescent reactions require molecular oxygen in the form ofdissolved or bound oxygen. Thus, molecular oxygen, dissolved in water orin air or bound to a photoprotein, is the activator for bioluminescencereactions. Depending upon the form of the components, other activatorsinclude, but are not limited to, ATP [for firefly luciferase], flavinreductase [bacterial systems] for regenerating FMNH₂ from FMN, and Ca²⁺or other suitable metal ion [aequorin].

Most of the systems provided herein will generate light when theluciferase and luciferin are mixed and exposed to air or water. Thesystems that use photoproteins that have bound oxygen, such as aequorin,however, will require exposure to Ca²⁺ [or other suitable metal ion],which can be provided in the form of an aqueous composition of a calciumsalt. In these instances, addition of a Ca²⁺ [or other suitable metalion] to a mixture of luciferase [aequorin] and luciferin [such ascoelenterazine] will result in generation of light. The Renilla systemand other Anthozoa systems also require Ca²⁺ [or other suitable metalion].

If crude preparations are used, such as ground up Cypridina [shrimp] orground fireflies, it may be necessary to add only water. In instances inwhich fireflies [or a firefly or beetle luciferase] are used thereaction may only require addition ATP. The precise components will beapparent, in light of the disclosure herein, to those of skill in thisart or may be readily determined empirically.

It is also understood that these mixtures will also contain anyadditional salts or buffers or ions that are necessary for each reactionto proceed. Since these reactions are well-characterized, those of skillin the art will be able to determine precise proportions and requisitecomponents. Selection of components will depend upon the apparatus,article of manufacture and luciferase. Various embodiments are describedand exemplified herein; in view of such description, other embodimentswill be apparent.

d. Reactions

In all embodiments, up to all but one component of a bioluminescencegenerating system will be mixed with or packaged with or otherwisecombined with a selected article of manufacture to produce the noveltyitem. When bioluminescence is desired, the remaining component(s) willbe added and light will be produced.

In general, since the result to be achieved is the production of lightvisible to the naked eye for entertainment, amusement or recreation, forthe purposes herein, the precise proportions and amounts of componentsof the bioluminescence reaction need not be stringently determined ormet. They must be sufficient to produce light. Generally, an amount ofluciferin and luciferase sufficient to generate a visible glow is used;this amount can be readily determined empirically and is dependent uponthe selected system and selected application.

For purposes herein, such amount is preferably at least theconcentrations and proportions used for analytical purposes by those ofskill in the such arts. Higher concentrations may be used if the glow isnot sufficiently bright. Also because the conditions in which thereactions are used are not laboratory conditions and the components aresubject to storage, higher concentration may be used to overcome anyloss of activity. Typically, the amounts are 1 mg, preferably 10 mg andmore preferably 100 mg, of a luciferase per liter of reaction mixture or1 mg, preferably 10 mg, more preferably 100 mg, coated on a portion of aT-shirt or other textile or paper. Such coating may be produced bydrying a composition containing at least about 0.01 mg/l, and typically0.1 mg/l, 1 mg/l, 10 mg/l or more of each component on the item. Theamount of luciferin is also between about 0.01 and 100 mg/l, preferablybetween 0.1 and 10 mg/l, additional luciferin can be added to many ofthe reactions to continue the reaction. In embodiments in which theluciferase acts catalytically and does not need to be regenerated, loweramounts of luciferase can be used. In those in which it is changedduring the reaction, it also can be replenished; typically higherconcentrations will be selected. Ranges of concentration per liter [orthe amount of coating on substrate the results from contacting with suchcomposition] of each component on the order of 0.1 to 20 mg, preferably0.1 to 10 mg, more preferably between about 1 and 10 mg of eachcomponent will be sufficient. When preparing coated substrates, asdescribed herein, greater amounts of coating compositions containinghigher concentrations of the luciferase or luciferin may be used.

Thus, for example, in presence of calcium, 5 mg of luciferin, such ascoelenterazine, in one liter of water will glow brightly for at leastabout 10 to 20 minutes, depending on the temperature of the water, whenabout 10 mgs of luciferase, such as aequorin photoprotein luciferase orluciferase from Renilla, is added thereto. Increasing the concentrationof luciferase, for example, to 100 mg/l, provides a particularlybrilliant display of light.

If desired, the onset of the bioluminescent reaction can be delayed byadding an, an inhibitor, for example magnesium, of the bioluminescencegenerating reaction. Also, where inhibition is not desired, theconcentration of free magnesium may be reduced by addition of asufficient amount of chelating agent, such asethylenediamine-tetraacetic acid [EDTA]. The amount of EDTA and alsocalcium can be empirically determined to appropriately chelatemagnesium, without inhibiting or preventing the desired bioluminescence.

It is understood, that concentrations and amounts to be used depend uponthe selected article of manufacture and they may be readily determinedempirically. Proportions, particularly those used when commencing anempirical determination, are generally those used for analyticalpurposes, and amounts or concentrations are at least those used foranalytical purposes, but the amounts can be increased, particularly if asustained and brighter glow is desired.

2. Ctenophore and Coelenterate Systems

Ctenophores, such as Mnemiopsis (mnemiopsin) and Beroe ovata (berovin),and coelenterates, such as Aequorea (aequorin), Obelia (obelin) andPelagia, produce bioluminescent light using similar chemistries [see,e.g., Stephenson et al. (1981) Biochimica et Biophysica Acta 678:65-75;Hart et al. (1979) Biochemistry 18:2204-2210; International PCTApplication No. WO94/18342, which is based on U.S. application Ser. No.08/017,116, U.S. Pat. No. 5,486,455 and other references and patentscited herein]. The Aequorin and Renilla systems are representative andare described in detail herein as exemplary and as among the presentlypreferred systems. The Aequorin and Renilla systems can use the sameluciferin and produce light using the same chemistry, but eachluciferase is different. The Aequorin luciferase aequorin, as well as,for example, the luciferases mnemiopsin and berovin, is a photoproteinthat includes bound oxygen and bound luciferin, requires Ca²⁺ [or othersuitable metal ion] to trigger the reaction, and must be regenerated forrepeated use; whereas, the Renilla luciferase acts as a true enzymebecause it is unchanged during the reaction and it requires dissolvedmolecular oxygen.

a. The Aequorin System

The aequorin system is well known [see, e.g., Tsuji et al. (1986)"Site-specific mutagenesis of the calcium-binding photoproteinaequorin," Proc. Natl. Acad. Sci. USA 83:8107-8111; Prasher et al.(1985) "Cloning and Expression of the cDNA Coding for Aequorin, aBioluminescent Calcium-Binding Protein," Biochemical and BiophysicalResearch Communications 126:1259-1268; Prasher et al. (1986) Methods inEnzymology 133:288-297; Prasher, et al. (1987) "Sequence Comparisons ofcDNAs Encoding for Aequorin Isotypes," Biochemistry 26:1326-1332;Charbonneau et al. (1985) "Amino Acid Sequence of the Calcium-DependentPhotoprotein Aequorin," Biochemistry 24:6762-6771; Shimomura et al.(1981) "Resistivity to denaturation of the apoprotein of aequorin andreconstitution of the luminescent photoprotein from the partiallydenatured apoprotein," Biochem. J. 199:825-828; Inouye et al. (1989) J.Biochem. 105:473-477; Inouye et al. (1986) "Expression of ApoaequorinComplementary DNA in Escherichia coli," Biochemistry 25:8425-8429;Inouye et al. (1985) "Cloning and sequence analysis of cDNA for theluminescent protein aequorin," Proc. Natl. Acad. Sci. USA 82:3154-3158;Prendergast, et al. (1978) "Chemical and Physical Properties of Aequorinand the Green Fluorescent Protein Isolated from Aequorea forskalea" J.Am. Chem. Soc. 17:3448-3453; European Patent Application 0 540 064 A1;European Patent Application 0 226 979 A2, European Patent Application 0245 093 A1 and European Patent Specification 0 245 093 B1; U.S. Pat. No.5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat.No. 5,422,266; U.S. Pat. No. 5,023,181; U.S. Pat. No. 5,162,227; and SEQID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form,described in U.S. Pat. No. 5,162,227, European Patent Application 0 540064 A1 and Sealite Sciences Technical Report No. 3 (1994), iscommercially available from Sealite, Sciences, Bogart, Ga. asAQUALITE®].

This system is among the preferred systems for use herein. As will beevident, since the aequorin photoprotein includes noncovalently boundluciferin and molecular oxygen, it is suitable for storage in this formas a lyophilized powder or encapsulated into a selected deliveryvehicle. The system can be encapsulated into pellets, such as liposomesor other delivery vehicles, or stored in single chamber dual or othermultiple chamber apparatus. When used, the vehicles are contacted with acomposition, even tap water, that contains Ca²⁺ [or other suitable metalion], to produce a mixture that glows. This system is preferred for usein numerous embodiments herein, such as in any embodiment that usespellets. These embodiments include, squirt guns, fairy dust, bubbletoys, bubble baths, soaps, linked to textiles, for addition to beveragesand foods.

(1) Aequorin and Related Photoproteins

The photoprotein, aequorin, isolated from the jellyfish, Aequorea, emitslight upon the addition of Ca²⁺ [or other suitable metal ion]. Theaequorin photoprotein, which includes bound luciferin and bound oxygenthat is released by Ca²⁺, does not require dissolved oxygen.Luminescence is triggered by calcium, which releases oxygen and theluciferin substrate producing apoaqueorin.

The bioluminescence photoprotein aequorin is isolated from a number ofspecies of the jellyfish Aequorea. It is a 22 kilodalton [kD] molecularweight peptide complex [see, e.g., Shimomura et al. (1962) J. Cellularand Comp. Physiol. 59:233-238; Shimomura et al. (1969) Biochemistry8:3991-3997; Kohama et al. (1971) Biochemistry 10:4149-4152; andShimomura et al. (1972) Biochemistry 11:1602-1608]. The native proteincontains oxygen and a heterocyclic compound coelenterazine, a luciferin,[see, below] noncovalently bound thereto. The protein contains threecalcium binding sites. Upon addition of trace amounts Ca²⁺ [or othersuitable metal ion, such as strontium] to the photoprotein, it undergoesa conformational change the catalyzes the oxidation of the boundcoelenterazine using the protein-bound oxygen. Energy from thisoxidation is released as a flash of blue light, centered at 469 nm.Concentrations of calcium ions as low as 10⁻⁶ M are sufficient totrigger the oxidation reaction.

Naturally-occurring apoaequorin is not a single compound but rather is amixture of microheterogeneous molecular species. Aequoria jellyfishextracts contain as many as twelve distinct variants of the protein[see, e.g., Prasher et al. (187) Biochemistry 26:1326-1332; Blinks etal. (1975) Fed. Proc. 34:474]. DNA encoding numerous forms has beenisolated [see, e.g., SEQ ID Nos. 5-9 and 13].

The photoprotein can be reconstituted [see, e.g., U.S. Pat. No.5,023,181] by combining the apoprotein, such as a protein recombinantlyproduced in E. coli, with a coelenterazine, such as a syntheticcoelenterazine, in the presence of oxygen and a reducing agent [see,e.g., Shimomura et al. (1975) Nature 256:236-238; Shimomura et al.(1981) Biochemistry J. 199:825-828], such as 2-mercaptoenthanol, andalso EDTA or EGTA [concentrations between about 5 to about 100 mM orhigher for applications herein] tie up any Ca²⁺ to prevent triggeringthe oxidation reaction until desired. DNA encoding a modified form ofthe apoprotein that does not require 2-mercaptoethanol forreconstitution is also available [see, e.g., U.S. Pat. No. U.S. Pat. No.5,093,240]. The reconstituted photoprotein is also commerciallyavailable [sold, e.g., under the trademark AQUALITE®, which is describedin U.S. Pat. No. 5,162,227].

The light reaction is triggered by adding Ca²⁺ at a concentrationsufficient to overcome the effects of the chelator and achieve the 10⁻⁶M concentration. Because such low concentrations of Ca²⁺ can trigger thereaction, for use in the methods and apparatus herein, higherconcentrations of chelator may be included in the compositions ofphotoprotein. Accordingly, higher concentrations of added Ca²⁺ in theform of a calcium salt will be required. Precise amounts may beempirically determined. For use herein, it may be sufficient to merelyadd water to the photoprotein, which is provided in the form of aconcentrated composition or in lyophilized or powdered form. Thus, forpurposes herein, addition of small quantities of Ca²⁺, such as thosepresent in most tap water or in phosphate buffered saline (PBS) or othersuitable buffers or possible in the moisture on the skin, should triggerthe bioluminescence reaction.

Numerous isoforms of the aequorin apoprotein been identified isolated.DNA encoding these proteins has been cloned, and the proteins andmodified forms thereof have been produced using suitable host cells[see, e.g., U.S. Pat. Nos. 5,162,227, 5,360,728, 5,093,240; see, also,Prasher et al. (1985) Biophys. Biochem. Res. Commun. 126:1259-1268;Inouye et al. (1986) Biochemistry 25: 8425-8429]. U.S. Pat. No.5,093,240; U.S. Pat. No. 5,360,728; U.S. Pat. No. 5,139,937; U.S. Pat.No. 5,288,623; U.S. Pat. No. 5,422,266, U.S. Pat. No. 5,162,227 and SEQID Nos. 5-13, which set forth DNA encoding the apoprotein; and a form iscommercially available form Sealite, Sciences, Bogart, Ga. asAQUALITE®]. DNA encoding apoaequorin or variants thereof is useful forrecombinant production of high quantities of the apoprotein. Thephotoprotein is reconstituted upon addition of the luciferin,coelenterazine, preferably a sulfated derivative thereof, or an analogthereof, and molecular oxygen [see, e.g., U.S. Pat. No. 5,023,181]. Theapoprotein and other constituents of the photoprotein andbioluminescence generating reaction can be mixed under appropriateconditions to regenerate the photoprotein and concomitantly have thephotoprotein produce light. Reconstitution requires the presence of areducing agent, such as mercaptoethanol, except for modified forms,discussed below, that are designed so that a reducing agent is notrequired [see, e.g., U.S. Pat. No. 5,093,240].

For use herein, it is preferred aequorin is produced using DNA, such asthat set forth in SEQ ID Nos. 5-13 and known to those of skill in theart or modified forms thereof. The DNA encoding aequorin is expressed ina host cell, such as E. coli, isolated and reconstituted to produce thephotoprotein [see, e.g., U.S. Pat. Nos. 5,418,155, 5,292,658, 5,360,728,5,422,266, 5,162,227].

Of interest herein, are forms of the apoprotein that have been modifiedso that the bioluminescent activity is greater than unmodifiedapoaequorin [see, e.g., U.S. Pat. No. 5,360,728, SEQ ID Nos. 10-12].Modified forms that exhibit greater bioluminescent activity thanunmodified apoaequorin include proteins having sequences set forth inSEQ ID Nos. 10-12, in which aspartate 124 is changed to serine,glutamate 135 is changed to serine, and glycine 129 is changed toalanine, respectively. Other modified forms with increasedbioluminescence are also available.

For use in certain embodiments herein, the apoprotein and othercomponents of the aequorin bioluminescence generating system arepackaged or provided as a mixture, which, when desired is subjected toconditions under which the photoprotein reconstitutes from theapoprotein, luciferin and oxygen [see, e.g., U.S. Pat. No. 5,023,181;and U.S. Pat. No. 5,093,240]. Particularly preferred are forms of theapoprotein that do not require a reducing agent, such as2-mercaptoethanol, for reconstitution. These forms, described, forexample in U.S. Patent No. 5,093,240 [see, also Tsuji et al. (1986)Proc. Natl. Acad. Sci. U.S.A. 83:8107-8111], are modified by replacementof one or more, preferably all three cysteine residues with, for exampleserine. Replacement may be effected by modification of the DNA encodingthe aequorin apoprotein, such as that set forth in SEQ ID No. 5, andreplacing the cysteine codons with serine.

The photoproteins and luciferases from related species, such as Obeliaare also contemplated for use herein. DNA encoding the Ca²⁺ -activatedphotoprotein obelin from the hydroid polyp Obelia longissima is knownand available [see, e.g., Illarionov et al. (1995) Gene 153:273-274; andBondar et al. (1995) Biochim. Biophys. Acta 1231:29-32]. Thisphotoprotein can also be activated by Mn²⁺ [see, e.g., Vysotski et al.(1995) Arch. Bioch. Biophys. 316:92-93, Vysotski et al. (1993) J.Biolumin. Chemilumin. 8:301-305].

In general for use herein, the components of the bioluminescence arepackaged or provided so that there is insufficient metal ions to triggerthe reaction. When used, the trace amounts of triggering metal ion,particularly Ca²⁺ is contacted with the other components. For a moresustained glow, aequorin can be continuously reconstituted or can beadded or can be provided in high excess.

(2) Luciferin

The aequorin luciferin is coelenterazine and analogs therein, whichinclude molecules having the structure [formula (I)]: ##STR1## in whichR₁ is CH₂ C₆ H₅ or CH₃ ; R₂ is C₆ H₅, and R₃ is p-C₆ H₄ OH or CH₃ orother such analogs that have activity. Preferred coelenterazine has thestructure in which R¹ is p-CH₂ C₆ H₄ OH, R₂ is C₆ H₅, and R₃ is p-C₆ H₄OH, which can be prepared by known methods [see, e.g., Inouye et al.(1975) Jap. Chem. Soc., Chemistry Lttrs. pp 141-144; and Hart et al.(1979) Biochemistry 18:2204-2210]. The preferred coelenterazine has thestructure (formula (II)): ##STR2## and sulfated derivatives thereof.

The reaction of coelenterazine when bound to the aequorin photoproteinwith bound oxygen and in the presence of Ca²⁺ can represented asfollows: ##STR3## The photoprotein aequorin [which contains apoaequorinbound to a coelenterate luciferin molecule] and Renilla luciferase,discussed below, can use the same coelenterate luciferin. The aequorinphotoprotein catalyses the oxidation of coelenterate luciferin[coelenterazine] to oxyluciferin [coelenteramide] with the concomitantproduction of blue light [lambda_(max) =469 nm].

Importantly, the sulfate derivative of the coelenterate luciferin[lauryl-luciferin] is particularly stable in water, and thus may be usedin a coelenterate-like bioluminescence generating system. In thissystem, adenosine diphosphate (ADP) and a sulpha-kinase are used toconvert the coelenterazine to the sulphated form. Sulfatase is then usedto reconvert the lauryl-luciferin to the native coelenterazine. Thus,the more stable lauryl-luciferin is used in the item to be illuminatedand the luciferase combined with the sulfatase are added to theluciferin mixture when illumination is desired.

Thus, the bioluminescence generating system of Aequorea is particularlysuitable for use in the methods and apparatus herein. The particularamounts and the manner in which the components are provided depends uponthe selected combination of article of manufacture. This system can beprovided in lyophilized form, that will glow upon addition of Ca²⁺. Itcan be encapsulated, linked to matrices, such as porous glass, or in asa compositions, such as a solution or suspension, preferably in thepresence of sufficient chelating agent to prevent triggering thereaction. The concentration of the aequorin photoprotein will vary andcan be determined empirically. Typically concentrations of at least 0.1mg/l, more preferably at least 1 mg/l and higher, will be selected. Incertain embodiments, 1-10 mg luciferin/100 mg of luciferase will be usedin selected volumes and at the desired concentrations will be used.

b. The Renilla System

Representative of coelenterate systems is the Renilla system. Renilla,also known as sea pansies, are members of the class of coelenteratesAnthozoa, which includes other bioluminescent genera, such asCavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus.Bioluminescent members of the Anthozoa genera contain luciferases andluciferins that are similar in structure [see, e.g., Cormier et al.(1973) J. Cell. Physiol. 81:291-298; see, also Ward et al. (1975) Proc.Natl. Acad. Sci. U.S.A. 72:2530-2534]. The luciferases and luciferinsfrom each of these anthozoans crossreact and produce a characteristicblue luminescence.

Renilla luciferase and the other coelenterate and ctenophoreluciferases, such as the aequorin photoprotein, use imidazopyrazinesubstrates, particularly the substrates generically calledcoelenterazine [see, formulae (I) and (II), above]. Other genera thathave luciferases that use a coelenterazine include: squid, such asChiroteuthis, Eucleoteuthis, Onychoteuthis, Watasenia; cuttlefish,Sepiolina; shrimp, such as Oplophorus, Sergestes, and Gnathophausia;deep-sea fish, such as Argyropelecus, Yarella, Diaphus, and Neoscopelus.

Renilla luciferase does not, however, have bound oxygen, and thusrequires dissolved oxygen in order to produce light in the presence of asuitable luciferin substrate. Since Renilla luciferase acts as a trueenzyme [i.e., it does not have to be reconstituted for further use] theresulting luminescence can be long-lasting in the presence of saturatinglevels of luciferin. Also, Renilla luciferase is relatively stable toheat.

Renilla luciferase, DNA encoding Renilla luciferase, and use of the DNAto produce recombinant luciferase, as well as DNA encoding luciferasefrom other coelenterates, are well known and available [see, e.g., SEQID No. 1, U.S. Pat. Nos. 5,418,155 and 5,292,658; see, also, Prasher etal. (1985) Biochem. Biophys. Res. Commun. 126:1259-1268; Cormier (1981)"Renilla and Aequorea bioluminescence" in Bioluminescence andChemiluminescence, pp. 225-233; Charbonneau et al. (1979) J. Biol. Chem.254:769-780; Ward et al. (1979) J. Biol. Chem. 254:781-788; Lorenz etal. (1981) Proc. Natl. Acad. Sci. U.S.A. 88: 4438-4442; Hori et al.(1977) Proc. Natl. Acad. Sci. U.S.A. 74:4285-4287; Hori et al. (1975)Biochemistry 14:2371-2376; Hori et al. (1977) Proc. Natl. Acad. Sci.U.S.A. 74:4285-4287; Inouye et al. (1975) Jap. Soc. Chem. Lett.141-144;and Matthews et al. (1979) Biochemistry 16:85-91]. The DNA encodingRenilla luciferase and host cells containing such DNA provide aconvenient means for producing large quantities of the enzyme [see,e.g., U.S. Pat. Nos. 5,418,155 and 5,292,658, which describe recombinantproduction of Renilla luciferase and the use of the DNA to isolate DNAencoding other luciferases, particularly those from related organisms].A modified version of a method [U.S. Pat. Nos. 5,418,155 and 5,292,658]for the recombinant production of Renilla luciferase that results in ahigher level of expression of the recombinant enzyme is presented in theEXAMPLES herein.

When used herein, the Renilla luciferase can be packaged, such as in antoy, in lyophilized form, encapsulated in a vehicle, either by itself orin combination with the luciferin substrate. Prior to use the mixture iscontacted with an aqueous composition, preferably a phosphate bufferedsaline or other suitable buffer, such a Tris-based buffer [such as 0.1mm Tris, 0.1 mm EDTA] pH 7-8, preferably about pH 8; dissolved O₂ willactivate the reaction. Addition of glycerol [about 1%] increases lightintensity. Final concentrations of luciferase in the glowing mixturewill be on the order of 0.01 to 1 mg/l or more. Concentrations ofluciferin will be at least about 10⁻⁸ M, but 1 to 100 or more orders ofmagnitude higher to produce a long lasting bioluminescence.

In certain embodiments herein, about 1 to 10 mg, or preferably 2-5 mg,more preferably about 3 mg of coelenterazine will be used with about 100mg of Renilla luciferase. The precise amounts, of course can bedetermined empirically, and, also will depend to some extent on theultimate concentration and application. In particular, about addition ofabout 0.25 ml of a crude extract from the bacteria that express Renillato 100 ml of a suitable assay buffer and about 0.005 μg was sufficientto produce a visible and lasting glow [see, U.S. Pat. Nos. 5,418,155 and5,292,658, which describe recombinant production of Renilla luciferase].

Lyophilized mixtures, and compositions containing the Renilla luciferaseare also provided. The luciferase or mixtures of the luciferase andluciferin may also be encapsulated into a suitable delivery vehicle,such as a liposome, glass particle, capillary tube, drug deliveryvehicle, gelatin, time release coating or other such vehicle. Kitscontaining these mixtures, compositions, or vehicles and also a selectedarticle of manufacture, such as a toy gun, bubble composition, balloon,item of clothing, personal item, are also provided. The luciferase mayalso be linked to a substrate, such as cotton, polyester,polyester-cotton blends, polypropylene, polyvinyltoluene, polyvinylpropylene, glass, ceramic, or plastics are provided in combination withor as part of an article of manufacture.

3. Crustacean, Particularly Cyrpidina Systems

The ostracods, such as Vargula serratta, hilgendorfii and noctiluca aresmall marine crustaceans, sometimes called sea fireflies. These seafireflies are found in the waters off the coast of Japan and emit lightby squirting luciferin and luciferase into the water, where thereaction, which produces a bright blue luminous cloud, occurs. Thereaction involves only luciferin, luciferase and molecular oxygen, and,thus, is very suitable for application herein.

The systems, such as the Vargula bioluminescence generating systems, areparticularly preferred herein because the components are stable at roomtemperature if dried and powdered and will continue to react even ifcontaminated. Further, the bioluminescent reaction requires only theluciferin/luciferase components in concentrations as low as 1:40 partsper billion to 1:100 parts per billion, water and molecular oxygen toproceed. An exhausted system can renewed by addition of luciferin.

a. Vargula luciferase

Vargula luciferase is a 555-amino acid polypeptide that has beenproduced by isolation from Vargula and also using recombinant technologyby expressing the DNA in suitable bacterial and mammalian host cells[see, e.g., Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6567-6571; Inouye et al. (1992) Proc. Natl. Acad. Sci. U.S.A.89:9584-9587; Johnson et al. (1978) Methods in Enzymology LVII:331-349;Tsuji et al. (1978) Methods Enzymol. 57:364-72; Tsuji (19740Biochemistry 13:5204-5209; Japanese Patent Application No. JP 3-30678Osaka; and European Patent Application No. EP 0 387 355 A1].

(1) Purification from Cypridina

Methods for purification of Vargula [Cypridina] luciferase are wellknown. For example, crude extracts containing the active can be readilyprepared by grinding up or crushing the Vargula shrimp. In otherembodiments, a preparation of Cypridina hilgendorfi luciferase can beprepared by immersing stored frozen C. hilgendorfi in distilled watercontaining, 0.5-5.0 M salt, preferably 0.5-2.0 M sodium or potassiumchloride, ammonium sulfate, at 0-30° C., preferably 0-10° C., for 1-48hr, preferably 10-24 hr, for extraction followed by hydrophobicchromatography and then ion exchange or affinity chromatography [TORAYIND INC, Japanese patent application JP 4258288, published Sep. 14,1993; see, also, Tsuji et al. (1978) Methods Enzymol. 57:364-72 forother methods].

The luciferin can be isolated from ground dried Vargula by heating theextract, which destroys the luciferase but leaves the luciferin intact[see, e.g., U.S. Pat. No. 4,853,327].

(2) Preparation by Recombinant Methods

The luciferase is preferably produced by expression of cloned DNAencoding the luciferase [European Patent Application NO. 0 387 355 A1;International PCT Application No. WO90/01542; see, also SEQ ID No. 5,which sets forth the sequence from Japanese Patent Application No. JP3-30678 and Thompson et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6567-6571] DNA encoding the luciferase or variants thereof isintroduced into E. coli using appropriate vectors and isolated usingstandard methods.

b. Vargula Luciferin

The natural luciferin in a substituted imidazopyrazine nucleus, such acompound of formula (III): ##STR4##

Analogs thereof and other compounds that react with the luciferase in alight producing reaction also may be used.

Other bioluminescent organisms that have luciferases that can react withthe Vargula luciferin include, the genera Apogon, Parapriacanthus andPorichthys.

c. Reaction

The luciferin upon reaction with oxygen forms a dioxetanone intermediate[which includes a cyclic peroxide similar to the firefly cyclic peroxidemolecule intermediate]. In the final step of the bioluminescentreaction, the peroxide breaks down to form CO₂ and an excited carbonyl.The excited molecule then emits a blue to blue-green light.

The optimum pH for the reaction is about 7. For purposes herein, any pHat which the reaction occurs may be used. The concentrations of reagentsare those normally used for analytical reactions or higher [see, e.g.,Thompson et al. (1990) Gene 96:257-262]. Typically concentrations of theluciferase between 0.1 and 10 mg/l, preferably 0.5 to 2.5 mg/l will beused. Similar concentrations or higher concentrations of the luciferinmay be used.

4. Insect Bioluminescence Generating Systems Including Firefly, ClickBeetle, and Other Insect Systems

The biochemistry of firefly bioluminescence was the firstbioluminescence generating system to be characterized [see, e.g.,Wienhausen et al. (1985) Photochemistry and Photobiology 42:609-611;McElroy et al. (1966) in Molecular Architecture in Cell Physiology,Hayashi et al., eds. Prentice Hall, Inc., Englewood Cliffs, N.J., pp.63-80] and it is commercially available [e.g., from Promega Corporation,Madison, Wis., see, e.g., Leach et al. (1986) Methods in Enzymology133:51-70, esp. Table 1]. Luciferases from different species offireflies are antigenically similar. These species include members ofthe genera Photinus, Photurins and Luciola. Further, the bioluminescentreaction produces more light at 30° C. than at 20° C., the luciferase isstabilized by small quantities of bovine albumin serum, and the reactioncan be buffered by tricine.

a. Luciferase

DNA clones encoding luciferases from various insects and the use toproduce the encoded luciferase is well known. For example, DNA clonesthat encode luciferase from Photinus pyralis, Luciola cruciata [see,e.g., de Wet et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:7870-7873;de We et al. (1986) Methods in Enzymology 133:3; U.S. Pat. No.4,968,613, see, also SEQ ID No. 3] are available. The DNA has also beenexpressed in Saccharomyces [see, e.g., Japanese Application No. JP63317079, published Dec. 26, 1988, KIKKOMAN CORP] and in tobacco.

In addition to the wild-type luciferase modified insect luciferases havebeen prepared. For example, heat stable luciferase mutants, DNA-encodingthe mutants, vectors and transformed cells for producing the luciferasesare available. A protein with 60% amino acid sequence homology withluciferases from Photinus pyralis, Luciola mingrelica, L. cruciata or L.lateralis and having luciferase activity is available [see, e.g.,International PCT Application No. WO95/25798]. It is more stable above30° C. than naturally-occurring insect luciferases and may also beproduced at 37° C. or above, with higher yield.

Modified luciferases that generate light at different wavelengths[compared with native luciferase], and thus, may be selected for theircolor-producing characteristics. For example, synthetic mutant beetleluciferase(s) and DNA encoding such luciferases that producebioluminescence at a wavelength different from wild-type luciferase areknown [Promega Corp, International PCT Application No. WO95/18853, whichis based on U.S. application Ser. No. 08/177,081 Jan. 3, 1994]. Themutant beetle luciferase has an amino acid sequence differing from thatof the corresponding wild-type Luciola cruciata [see, e.g., U.S. Pat.Nos. 5,182,202, 5,219,737, 5,352,598, see, also SEQ ID No. 3] by asubstitution(s) at one or two positions. The mutant luciferase producesa bioluminescence with a wavelength of peak intensity that differs by atleast 1 nm from that produced by wild-type luciferases.

Other mutant luciferase have also been produced. Mutant luciferases withthe amino acid sequence of wild-type luciferase, but with at least onemutation in which valine is replaced by isoleucine at the amino acidnumber 233, valine by isoleucine at 239, serine by asparagine at 286,glycine by serine at 326, histidine by tyrosine at 433 or proline byserine at 452 are known [see, e.g., U.S. Pat. Nos. 5,219,737, and5,330,906]. The luciferases are produced by expressing DNA-encoding eachmutant luciferase in E. coli and isolating the protein. Theseluciferases produce light with colors that differ from wild-type. Themutant luciferases catalyze luciferin to produce red [λ 609 nm and 612nm], orange [λ595 and 607 nm] or green [λ 558 nm] light. The otherphysical and chemical properties of mutant luciferase are substantiallyidentical to native wild type-luciferase. The mutant luciferase has theamino acid sequence of Luciola cruciata luciferase with an alterationselected from Ser 286 replaced by Asn, Gly 326 replaced by Ser, His 433replaced by Tyr or Pro 452 replaced by Ser. Thermostable luciferases arealso available [see, e.g., U.S. Pat. No. 5,229,285; see, alsoInternational PCT Application No. WO95/25798, which provides Photinusluciferase in which the glutamate at position 354 is replaced lysine andLuciola luciferase in which the glutamate at 356 is replaced withlysine].

These mutant luciferases as well as the wild type luciferases are amongthose preferred herein, particularly in instances when a variety ofcolors are desired or when stability at higher temperatures is desired.It is also noteworthy that firefly luciferases have alkaline pH optima[7.5-9.5], and, thus, are suitable for use in combination with articlesof manufacture, such as the bubble compositions that have alkaline pH.

b. Luciferin

The firefly luciferin is a benzothiazole: ##STR5## Analogs of thisluciferin and synthetic firefly luciferins are also known to those ofskill in art [see, e.g., U.S. Pat. Nos. 5,374,534 and 5,098,828]. Theseinclude compounds of formula (IV) [see, U.S. Pat. No. 5,098,828]:##STR6## in which: R¹ is hydroxy, amino, linear or branched C₁ -C₂₀alkoxy, C₂ -C₂₀ alkyenyloxy, an L-amino acid radical bond via theα-amino group, an oligopeptide radical with up to ten L-amino acid unitslinked via the α-amino group of the terminal unit;

R² is hydrogen, H₂ PO₃, HSO₃, unsubstituted or phenyl substituted linearor branched C₁ -C₂₀ alkyl or C₂ -C₂₀ alkenyl, aryl containing 6 to 18carbon atoms, or R³ --C(O)--; and

R³ is an unsubstituted or phenyl substituted linear or branched C₁ -C₂₀alkyl or C₂ -C₂₀ alkenyl, aryl containing 6 to 18 carbon atoms, anucleotide radical with 1 to 3 phosphate groups, or a glycosidicallyattached mono- or disaccharide, except when formula (IV) is aD-luciferin or D-luciferin methyl ester.

c. Reaction

The reaction catalyzed by firefly luciferases and related insectluciferases requires ATP, Mg²⁺ as well as molecular oxygen. Luciferinmust be added exogenously. Firefly luciferase catalyzes the fireflyluciferin activation and the subsequent steps leading to the excitedproduct. The luciferin reacts with ATP to form a luciferyl adenylateintermediate. This intermediate then reacts with oxygen to form a cyclicluciferyl peroxy species, similar to that of the coelenterateintermediate cyclic peroxide, which breaks down to yield CO₂ and anexcited state of the carbonyl product. The excited molecule then emits ayellow light; the color, however, is a function of pH. As the pH islowered the color of the bioluminescence changes from yellow-green tored.

Different species of fireflies emit different colors of bioluminescenceso that the color of the reaction will be dependent upon the speciesfrom which the luciferase is obtained. Additionally, the reaction isoptimized at pH 7.8.

Addition of ATP and luciferin to a reaction that is exhausted producesadditional light emission. Thus, the system, once established, isrelatively easily maintained. Therefore, it is highly suitable for useherein in embodiments in which a sustained glow is desired or reuse ofthe item is contemplated. Thus, the components of a firefly system canbe packaged with the item of manufacture, such as a toy gun, and thencombined with the article before use. For example, the luciferin and ATPcan be added to a mild bubble or a protein composition that containsluciferase each time the bubbles are used.

5. Bacterial Systems

Luminous bacteria typically emit a continuous light, usually blue-green.When strongly expressed, a single bacterium may emit 10⁴ to 10⁵ photonsper second. Bacterial bioluminescence systems include, among others,those systems found in the bioluminescent species of the generaPhotobacterium, Vibrio and Xenorhabdus. These systems are well known andwell characterized [see, e.g., Baldwin et al. (1984) Biochemistry23:3663-3667; Nicoli et al. (1974) J. Biol. Chem. 249:2393-2396; Welcheset al. (1981) Biochemistry 20:512-517; Engebrecht et al. (1986) Methodsin Enzymology 133:83-99; Frackman et al. (1990) J. of Bacteriology172:5767-5773; Miyamoto et al. (1986) Methods in Enzymology 133:70; U.S.Pat. No. 4,581,335].

a. Luciferases

Bacterial luciferase, as exemplified by luciferase derived from Vibrioharveyi [EC 1.14.14.3, alkanol reduced-FMN-oxygen oxidoreductase1-hydroxylating, luminescing], is a mixed function oxidase, formed bythe association of two different protein subunits α and β. The α-subunithas an apparent molecular weight of approximately 42,000 kD and theβ-subunit has an apparent molecular weight of approximately 37,000 kD[see, e.g., Cohn et al. (1983) Proc. Natl. Acad. Sci. U.S.A.80:120-123]. These subunits associate to form a 2-chain complexluciferase enzyme, which catalyzes the light emitting reaction ofbioluminescent bacteria, such as Vibrio harveyi [U.S. Pat. No.4,581,335; Belas et al. (1982) Science 218:791-793], Vibrio fischeri[Engebrecht et al. (1983) Cell 32:773-781; Engebrecht et al. (1984)Proc. Natl. Acad. Sci. U.S.A. 81:4154-4158] and other marine bacteria.

Bacterial luciferase genes have been cloned [see, e.g., U.S. Pat. No.5,221,623; U.S. Pat. No. 4,581,335; European Patent Application No. EP386 691 A]. Plasmids for expression of bacterial luciferase, such asVibrio harveyi, include pFIT001 (NRRL B-18080), pPALE001 (NRRL B-18082)and pMR19 (NRRL B-18081)] are known. For example the sequence of theentire lux regulon from Vibiro fisheri has been determined [Baldwin etal. (1984), Biochemistry 23:3663-3667; Baldwin et al. (1981) Biochem.20: 512-517; Baldwin et al. (1984) Biochem. 233663-3667; see, also,e.g., U.S. Pat. Nos. 5,196,318, 5,221,623, and 4,581,335]. This regulonincludes luxl gene, which encodes a protein required for autoinducersynthesis [see, e.g., Engebrecht et al. (1984) Proc. Natl. Acad. Sci.U.S.A. 81:4154-4158], the luxC, luxD, and luxE genes, which encodeenzymes that provide the luciferase with an aldehyde substrate, and theluxA and luxB genes, which encode the alpha and beta subunits of theluciferase.

Lux genes from other bacteria have also been cloned and are available[see, e.g., Cohn et al. (1985) J. Biol. Chem. 260:6139-6146; U.S. Pat.No. 5,196,524, which provides a fusion of the luxA and luxB genes fromVibrio harveyi]. Thus, luciferase alpha and beta subunit-encoding DNA isprovided and can be used to produce the luciferase. DNA encoding the α[1065 bp] and β [984 bp] subunits, DNA encoding a luciferase gene of2124 bp, encoding the alpha and beta subunits, a recombinant vectorcontaining DNA encoding both subunits and a transformed E. coli andother bacterial hosts for expression and production of the encodedluciferase are available. In addition, bacterial luciferases arecommercially available.

b. Luciferins

Bacterial luciferins include: ##STR7## in which the tetradecanal withreduced flavin mononucleotide are considered luciferin since both areoxidized during the light emitting reaction.

c. Reactions

The bacterial systems require, in addition to reduced flavin, fivepolypeptides to complete the bioluminescent reaction: two subunits, αand β, of bacterial luciferin and three units of a fatty acid reductase-system complex, which supplies the tetradecanal aldehyde. Examples ofbacterial bioluminescence generating systems useful in the apparatus andmethods provided herein include those derived from Vibrio fisheri andVibrio harveyi. One advantage to this system is its ability to operateat cold temperatures. It will thus be particularly amenable to use inice cubes. All components of a bacterial system can be frozen into icecubes. As it the ice cubes melt into a warmer beverage, which hasdissolved O₂, the reaction will proceed, thereby providing a sustainedglow.

Bacterial luciferase catalyzes the flavin-mediated hydroxylation of along-chain aldehyde to yield carboxylic acid and an excited flavin; theflavin decays to ground state with the concomitant emission of bluegreen light [λ_(max) =490 nm; see, e.g., Legocki et al. (1986) Proc.Natl. Acad. Sci. USA 81:9080; see U.S. Pat. No. 5,196,524]: ##STR8## Thereaction can be initiated by contacting reduced flavin mononucleotide[FMNH₂ ] with a mixture of the bacterial luciferase, oxygen, and along-chain aldehyde, usually n-decyl aldehyde.

DNA encoding luciferase from the fluorescent bacterium Alteromonashanedai is known [CHISSO CORP; see, also, Japanese application JP7222590, published Aug. 22, 1995]. The reduced flavin mononucleotide[FMNH2; luciferin] reacts with oxygen in the presence of bacterialluciferase to produce an intermediate peroxy flavin. This intermediatereacts with a long-chain aldehyde [tetradecanal] to form the acid andthe luciferase-bound hydroxy flavin in its excited state. The excitedluciferase-bound hydroxy flavin then emits light and dissociates fromthe luciferase as the oxidized flavin mononucleotide [FMN] and water. Invivo FMN is reduced again and recycled, and the aldehyde is regeneratedfrom the acid.

Flavin reductases have been cloned [see, e.g., U.S. Pat. No. 5,484,723;see, SEQ ID No. 14 for a representative sequence from this patent].These as well as NAD(P)H can be included in the reaction to regenerateFMNH₂ for reaction with the bacterial luciferase and long chainaldehyde. The flavin reductase catalyzes the reaction of FMN, which isthe luciferase reaction, into FMNH₂ ; thus, if luciferase and thereductase are included in the reaction system, it is possible tomaintain the bioluminescent reaction. Namely, since the bacterialluciferase turns over many times, bioluminescence continues as long as along chain aldehyde is present in the reaction system.

The color of light produced by bioluminescent bacteria also results fromthe participation of a protein blue-florescent protein [BFP] in thebioluminescence reaction. This protein, which is well known [see, e.g.,Lee et al. (1978) Methods in Enzymology LVII:226-234], and may also beadded to bacterial bioluminescence reactions in order to cause a shiftin the color.

6. Other Systems

a. Dinoflagellate Bioluminescence generating Systems

In dinoflagellates, bioluminescence occurs in organelles termedscintillons. These organelles are outpocketings of the cytoplasm intothe cell vacuole. The scintillons contain only dinoflagellate luciferaseand luciferin [with its binding protein], other cytoplasmic componentsbeing somehow excluded. The dinoflagellate luciferin is a tetrapyrrolerelated to chlorophyll: ##STR9## or an analog thereof.

The luciferase is a 135 kD single chain protein that is active at pH6.5, but inactive at pH 8 [see, e.g., Hastings (1981) Bioluminescenceand Chemiluminescence, DeLuca et al., eds. Academic Press, NY,pp.343-360]. Luminescent activity can be obtained in extracts made at pH8 by shifting the pH from 8 to 6. This occurs in soluble and particulatefractions. Within the intact scintillon, the luminescent flash occursfor ˜100 msec, which is the duration of the flash in vivo. In solution,the kinetics are dependent on dilution, as in any enzymatic reaction. AtpH 8, the luciferin is bound to a protein [luciferin binding protein]that prevents reaction of the luciferin with the luciferase. At pH 6,however, the luciferin is released and free to react with the enzyme.

b. Systems from Molluscs, Such as Latia and Pholas

Molluscs Latia neritoides and species of Pholas are bioluminescentanimals. The luciferin has the structure: ##STR10## and has beensynthesized [see, e.g., Shimomura et al. (1968) Biochemistry7:1734-1738; Shimomura et al. (1972) Proc. Natl. Acad. Sci. U.S.A.69:2086-2089]. In addition to a luciferase and luciferin the reactionhas a third component, a "purple protein". The reaction, which can beinitiated by an exogenous reducing agent is represented by the followingscheme: ##STR11## XH₂ is a reducing agent.

Thus for practice herein, the reaction will require the purple proteinas well as a reducing agent.

c. Earthworms and Other Annelids

Earthworm species, such as Diplocardia longa, Chaetopterus andHarmothoe, exhibit bioluminescence. The luciferin has the structure:##STR12##

The reaction requires hydrogen peroxide in addition to luciferin andluciferase. The luciferase is a photoprotein.

d. Glow Worms

The luciferase/luciferin system from the glow worms that are found inNew Zealand caves, Australia and those found in Great Britain are alsointended for use herein.

e. Marine Polycheate Worm Systems

Marine polycheate worm bioluminescence generating systems, such asPhyxotrix and Chaetopterus, are also contemplated for use herein.

f. South American Railway Beetle

The bioluminescence generating system from the South American railwaybeetle is also intended for use herein.

g. Fish

Of interest herein, are luciferases and bioluminescence generatingsystems that generate red light. These include luciferases found inspecies of Aristostomias, such as A. scintillans [see, e.g., O'Day etal. (1974) Vision Res. 14:545-550], Pachystomias, Malacosteus, such asM. niger.

7. Fluorescent Proteins

Fluorescent proteins (FPs), particularly green fluorescent proteins(GFPs), such as those from Aquorea and Renilla, and other relatedproteins can be used in combination with any of the novelty itemsprovided herein, including toys, beverages, foods, cosmetics, paperproducts and others. The FPs may be used alone with these items or maybe added to bioluminescence generating systems or items with suchsystems as a means of altering the color of the items. Mutein GFPs fromAquorea are also known (see, e.g., U.S. Pat. No. 5,625,048).

a. Green and Blue Fluorescent Proteins

Blue light is produced using the Renilla luciferase or the Aequoreaphotoprotein in the presence of Ca²⁺ and the coelenterazine luciferin oranalog thereof. This light can be converted into a green light if agreen fluorescent protein (GFP) is added to the reaction. Greenfluorescent proteins, which have been purified [see, e.g., Prasher etal. (1992) Gene 111:229-233] and also cloned [see, e.g., InternationalPCT Application No. WO 95/07463, which is based on U.S. application Ser.No. 08/119,678 and U.S. application Ser. No. 08/192,274, which areherein incorporated by reference], are energy-transfer acceptors. GFPsfluoresce in vivo upon receiving energy from a luciferase-oxyluciferinexcited-state complex or a Ca²⁺ -activated photoprotein. The chromophoreis formed from modified amino acid residues within the polypeptide.

The best characterized GFPs are those of Aequorea and Renilla [see,e.g., Prasher et al. (1992) Gene 111 :229-233; Hart, et al. (1979)Biochemistry 18:2204-2210]. For example, a green fluorescent protein[GFP] from Aequorea victoria contains 238 amino acids, absorbs bluelight and emits green light.

Thus, for example, inclusion of this protein in a composition containingthe aequorin photoprotein charged with coelenterazine and oxygen, can,in the presence of calcium, result in the production of green light. Itis contemplated that GFPs may be included in the bioluminescencegenerating reactions that employ the aequorin or Renilla luciferases orother suitable luciferase in order to enhance or alter color of theresulting bioluminescence.

GFPs are activated by blue light to emit green light and thus may beused in the absence of luciferase and in conjunction with an externallight source with novelty items, as described herein. Similarly, bluefluorescent proteins (BFPs), such as from Vibrio fischeri, Vibrioharveyi or Photobacterium phosphoreum, may be used in conjunction withan external light source of appropriate wavelength to generate bluelight. (See for example, Karatani, et al., "A blue fluorescent proteinfrom a yellow-emitting luminous bacterium," Photochem. Photobiol.55(2):293-299 (1992); Lee, et al., "Purification of a blue-fluorescentprotein from the bioluminescent bacterium Photobacterium phosphoreum"Methods Enzymol. (Biolumin. Chemilumin.) 57:226-234 (1978); and Gast, etal. "Separation of a blue fluorescence protein from bacterialluciferase" Biochem. Biophys. Res. Commun. 80(1):14-21 (1978), each, asall references cited herein, incorporated in its entirety by referenceherein.) In particular, GFPs, and/or BFPs or other such fluorescentproteins may be used in the beverage and/or food combinations providedherein and served in rooms illuminated with light of an appropriatewavelength to cause the fluorescent proteins to fluoresce.

GFPs and/or BFPs or other such fluorescent proteins may be used in anyof the novelty items and combinations provided herein, such as thebeverages and toys, including bubble making toys, particularlybubble-making compositions or mixtures and cosmetics. Such systems areparticularly of interest because no luciferase is needed to activate thephotoprotein and because the proteins are readily digested. Thesefluorescent proteins may also be used in addition to bioluminescencegenerating systems to enhance or create an array of different colors.

These proteins may be used alone or in combination with bioluminescencegenerating systems to produce an array of colors. They may be used incombinations such that the color of, for example, a beverage changesover time, or includes layers of different colors.

b. Phycobiliproteins

Phycobiliproteins are water soluble fluorescent proteins derived fromcyanobacteria and eukaryotic algae [see, e.g., Apt et al. (1995) J. Mol.Biol. 238:79-96; Glazer (1982) Ann. Rev. Microbiol. 36:173-198; andFairchild et al. (1994) J. of Biol. Chem. 269:8686-8694]. These proteinshave been used as fluorescent labels in immunoassay [see, Kronick (1986)J. of Immunolog. Meth. 92:1-13], the proteins have been isolated and DNAencoding them is also available [see, e.g., Pilot et al. (1984) Proc.Natl. Acad. Sci. U.S.A. 81:6983-6987; Lui et al. (1993) Plant Physiol103:293-294; and Houmard et al. (1988) J. Bacteriol. 170:5512-5521; theproteins are commercially available from, for example, ProZyme, Inc.,San Leandro, Calif.].

In these organisms, the phycobiliproteins are arranged in subcellularstructures termed phycobilisomes and function as accessory pigments thatparticipate in photosynthetic reactions by absorbing visible light andtransferring the derived energy to chlorophyll via a direct fluorescenceenergy transfer mechanism.

Two classes of phycobiliproteins are known based on their color:phycoerythrins (red) and phycocyanins (blue), which have reportedabsorption maxima between 490 and 570 nm and between 610 and 665 nm,respectively. Phycoerythrins and phycocyanins are heterogenous complexescomposed of different ratios of alpha and beta monomers to which one ormore class of linear tetrapyrrole chromophores are covalently bound.Particular phycobiliproteins may also contain a third γ-subunit whichoften associated with (αβ)₆ aggregate proteins.

All phycobiliproteins contain either phycothrombilin or phycoerythobilinchromophores, and may also contain other bilins, such as phycourobilin,cryptoviolin or a 697 nm bilin. The γ-subunit is covalently bound withphycourobilin, which results in the 495-500 nm absorbance peak of B- andR-phycoerythrins. Thus, the spectral characteristics ofphycobiliproteins may be influenced by the combination of the differentchromophores, the subunit composition of the apophycobiliproteins and/orthe local environment that affects the tertiary and quaternary structureof the phycobiliproteins.

As described above for GFPs & BFPs, phycobiliproteins are also activatedby visible light of the appropriate wavelength and thus may be used inthe absence of luciferase and in conjunction with an external lightsource to illuminate novelty items, particularly, as described herein.In particular, phycobiliproteins may be used in the novelty items, suchas beverage and/or food combinations provided herein and served in roomsilluminated with light of an appropriate wavelength to cause thefluorescent proteins to fluoresce. Cosmetics containing these proteinsare also contemplated.

As noted above, these proteins may be used in combination with otherfluorescent proteins and/or bioluminescence generating systems toproduce an array of colors or to provide different colors over time.

Attachment of phycobiliproteins to solid support matrices is known(e.g., see U.S. Pat. Nos. 4,714,682; 4,767,206; 4,774,189 and4,867,908). Therefore, phycobiliproteins may be coupled to microcarrierscoupled to one or more components of the bioluminescent reaction,preferably a luciferase, to convert the wavelength of the lightgenerated from the bioluminescent reaction. Microcarriers coupled to oneor more phycobiliproteins may be used in any of the novelty items andcombinations provided herein, such as the multicolor beverages and toys,including bubble making toys, particularly bubble-making compositions ormixtures.

C. Practice of the Reactions in Combination with Articles of Manufacture

The particular manner in which each bioluminescence system will becombined with a selected article of manufacture will be a function ofthe article and the desired effect. In general, however, less than allof the components of the reaction will be provided with the article andthen contact with the remaining component(s) to produce a glow. Thereare a multitude of alternative means for achieving this result; some aredescribed herein, and others will be apparent by virtue of thedisclosure herein.

In the simplest embodiments, the organisms can be ground up and dried.For example, light will be emitted by ground up fireflies when mixedwith water and ATP. Light will also be emitted merely be combiningground up Vargula shrimp and adding water, preferably cool water [roomtemperature or lower]. The only caveat is that the water must not be toohot; high temperatures destroy activity of the luciferases.

In other embodiments, the substantially pure reagents are combined withthe article of manufacture and the article will glow or spew a glowingspray or jet. The reagents may be provided in compositions, such assuspensions, as powders, as pastes or any in other suitable form. Theymay be provided as sprays, aerosols, or in any suitable form. Thereagents may be linked to a matrix and combined with the article ofmanufacture or formed into the article of manufacture. Typically all butone or more, though preferably all but one, of the components necessaryfor the reaction will be mixed and provided together; reaction will betriggered contacting the mixed component(s) with the remainingcomponent(s), such as by adding Ca²⁺, FMN with reductase, FMNH₂, ATP,air or oxygen. The resulting matrix materials are advantageously used inconnection numerous novelty items, such as clothing. They are also usedin the cartridges provided herein.

In preferred embodiments the luciferase or luciferase/luciferin, such asthe aequorin photoprotein, will be provided in combination with thearticle of manufacture or added before use. The article will then becontacted with the remaining components. As will become apparent herein,there are a multitude of ways in which each system may be combined witha selected article of manufacture.

D. Packaging of Bioluminescence Systems

Packaging for bioluminescence generating reagents provided herein mustbe chosen according to the article of manufacture with which thereagents are to be combined. In general, the packaging is non-reactivewith the compositions contained therein and must exclude water and orair to the degree those substances are required for the luminescentreaction to proceed. It will be appreciated, however, that specific usesfor the bioluminescence generating systems may require specificpackaging. Following are some examples of the special packagingrequirements of various end uses of the bioluminescence generatingsystems. These are offered as examples only and are in no way intendedas limiting.

The bioluminescence generating reagents may be provided in pellets,encapsulated as micro or macro-capsules, linked to matrices and includedin or on articles of manufacture, or as mixtures in chambers within anarticle of manufacture or in some other configuration. With respect toother articles of manufacture that include chambers or vessels, such ascertain toys, primary considerations are that the bioluminescencegenerating system be amenable to activation by the user at will and thatthe container be non-reactive and, if desired, translucent to thebioluminescent glow. Examples of vessels include beverage holders,plates or other dishes, vases, jars, bottles, spray cans and othercontainers. In general, vessels for use in practicing the methods hereinhave an enclosed, defined space, that contains most of the components ofthe bioluminescence generating system, and a separate enclosed, definedspace containing the remaining necessary ingredients; such that, the twospaces are separated by a readily removable membrane which, uponremoval, permits the components to mix and thereby react, resulting inillumination. Alternatively, the vessel can have a single compartmentcontaining all but the final ingredients of the bioluminescencegenerating system and being amenable to addition of the finalingredients by the user; for example through an opening in thecompartment.

Any toy, vessel or other article of manufacture that is amenable tohaving a generally translucent covering defining a space for containmentof the bioluminescence generating system components and that is amenableto simple manipulation to permit addition of the final componentsnecessary for the illumination reaction is contemplated.

Thus, whether the item that will glow or produce a glowing fluid, jet orspray, is a toy, vessel or other article of manufacture, its generaldesign is the same. At least one of the bioluminescence generatingsystem components is separated from the remaining components. Theremaining components are added prior to use. They can be included in thearticle of manufacture and physically separated from the othercomponents. For example, the physical separation means are those thatare readily removed by the user, to permit mixing, resulting inillumination of the components. For example, an article of manufacturemay contain a luciferase and substrate in one compartment and abioluminescence activator in an adjacent compartment; or alternatively,one compartment may contain the luciferase, and the other the substrateluciferin and dissolved oxygen or other requisite activator(s). Thecompartments are separated by a dividing member, such as a membrane,that, upon compression of the article of manufacture, rupturespermitting separated components to mix and to thereby glow. For suitableembodiments, see EXAMPLES, below [see, also, e.g., containers describedin U.S. Pat. Nos. 3,539,794 and 5,171,081].

Other embodiments contemplated herein, include those in which a fluid isejected as a spray or jet and is rendered bioluminescent prior toejection from the device, such as a toy or fountain. In general, themethods will involve addition of the bioluminescence generating systemcomponents to the water just prior to ejection thereby causing theejected spray or jet or stream to glow. Various apparatus foraccomplishing this are provided herein. In light of the disclosureherein other apparatus can be adapted for such use. Examples includechambers within a toy that inject the components into a water chamberjust prior to ejection of the water, or a clip-on device housing thecomponents, perhaps in premeasured amounts, which is attached to the toyand manually or automatically engaged to inject the ingredients into awater chamber. Similarly, the water can be introduced into a chambercontaining the components and then ejected.

In other embodiments, the components may be packaged as separatecompositions, that, upon mixing, glow. For example, a compositioncontaining luciferase may be provided separately from, and for use with,an a separate composition containing a bioluminescence substrate andbioluminescence activator. In another instance, luciferase and luciferincompositions may be separately provided and the bioluminescenceactivator may be added after, or simultaneously with, mixing of theother two compositions.

Similarly, the luciferase and bioluminescence substrate may be providedin a single packaging apparatus, an composition that is a mixture,suspension, solution, powder, paste or other suitable composition, thatis designed to exclude the necessary bioluminescence activator. Uponaddition of the bioluminescence activator to the remaining components orupon addition of the components to the bioluminescence activator, thereaction commences and the mixture glows. One example of such a systemis "fairy dust". In this embodiment the luciferase and bioluminescencesubstrate, for example, are packaged to exclude water and/or air, thebioluminescence activator. Release of the components from the packaginginto the air and/or moisture in the air activates the components therebygenerating luminescence. Another example is packaging the luciferase andsubstrate in the cap apparatus of a vessel, such that operation of thecap apparatus releases the components into the composition contained inthe vessel, causing it to glow.

1. Dispensing and Packaging Apparatus for Combination with theBioluminescence generating system Components

In one aspect, the bioluminescent apparatus systems provided herein arebioluminescence [or bioluminescent] systems in combination withdispensing or packaging apparatus. The bioluminescence systems,described in detail elsewhere herein, include three components: abioluminescence substrate [e.g., a luciferin], a luciferase [e.g., aluciferase or photoprotein], and a bioluminescence activator oractivators [e.g., molecular oxygen or Ca²⁺ ]. The dispensing andpackaging apparatus are configured to keep at least one of the threecomponents separate from the other two components, until generation ofbioluminescence is desired.

In general, the dispensing and packaging apparatus are non-reactive withthe bioluminescence generating system components contained therein andcan exclude moisture, air or other activators, such as O₂ or Ca²⁺, or insome manner keep all necessary components that are required for thebioluminescent reaction to come into contact until desired.

It will be appreciated, however, that specific applications andconfigurations of the bioluminescence systems may require specificapparatus. Following are exemplary descriptions of various dispensersand packages contemplated for use herein. These are offered as examplesonly and are in no way intended as limiting. It is understood that inlight of the description herein, other apparatus may be modified ordevised, that would be suitable for use to produce bioluminescence incombination with novelty items.

2. Capsules, Pellets, Liposomes, Endosomes, Vacuoles, MicronizedParticles

Certain embodiments of the novelty item combinations provided hereinrequire sequestering of the components from the environment prior to useor require the components to be provided in particulate form. Examplesof such embodiments include beverages, foods and particles, such as foruse as fairy dust or in toy guns, fountains of particles and other suchapplications. In particular, embodiments in which the bioluminescencegenerating system is manufactured as part of food or beverage producingglowing beverages or foods require specific packaging considerations. Tobe amenable to use as an additive to beverages for human consumption,the packaging must be non-toxic, and should be easy to open to providefor contact of the bioluminescence generating system components with thebeverage. Examples of suitable packaging for such use includeencapsulating the bioluminescence generating system components in one ormicro- [up to about 100 μm in size] or macroparticles [larger than 100μM] of material that permits release of the contents, such as bydiffusion or by dissolution of the encapsulating material. Liposomes andother encapsulating vehicles [see, e.g., U.S. Pat. No. 4,525,306, whichdescribes encapsulation of compounds in gelatin; U.S. Pat. Nos.4,021,364, 4,225,581, 4,269,821, 4,322,311, 4,324,683, 4,329,332,4,525,306, 4,963,368 describe encapsulation of biologically activematerials in various polymers] known to those of skill in the art,including those discussed herein and known to those of skill in the art[such as soluble paper, see U.S. Pat. No. 3,859,125]. Likewise,packaging of the system components for addition to food products mustaddress the same considerations. The components may be added to the foodsubstance directly, e.g., by sprinkling the dried and powderedingredients onto the food, or indirectly, e.g., via addition, to thefood, of a capsule containing the ingredients.

a. Encapsulating Vehicles in General

All components of the bioluminescence generating system, except for theoxygen or water or Ca²⁺, depending upon the selected system can beincorporated into encapsulating material, such as liposomes, thatprotect the contents from the environment until placed into conditionsthat cause release of the contents into the environment. Encapsulatingmaterial contemplated for use herein includes liposomes and other suchmaterials used for encapsulating chemicals, such as drug deliveryvehicles.

b. Encapsulating Vehicles--Liposomes

For example, liposomes that dissolve and slowly release the componentsinto the selected beverage, which contains dissolved oxygen or Ca²⁺ oreven ATP for the luciferase system are contemplated herein. They can beformulated in compositions, such as solutions, suspensions, gels,lotions, creams, and ointments. Liposomes and other slow releaseencapsulating compositions are well known and can be adapted for use infor slow release delivery of bioluminescence generating components.Typically the luciferin and luciferase will be encapsulated in theabsence of oxygen or Ca²⁺ or ATP or other activating component. Uponrelease into the environment or medium containing this component at asuitable concentration, the reaction will proceed and a glow will beproduced. Generally the concentrations of encapsulated components shouldbe relatively high, perhaps 0.1-1 mg/ml or more, to ensure high enoughlocal concentrations upon release to be visible.

Liposomes or other sustained release delivery system that are formulatedin an ointment or sustained release topical vehicle, for example, wouldbe suitable for use in a body paint, lotion. Those formulated as asuspension would be useful as a spray. Numerous ointments and suitableliposome formulations are known [see, e.g., Liposome Technology,Targeted Drug Delivery and Biological Interaction, vol. III, G.Gregoriadis ed., CRC Press, Inc., 1984; U.S. Pat. Nos. 5,470,881;5,366,881; 5,296,231; 5,272,079; 5,225,212; 5,190,762; 5,188,837;5,188,837; 4,921,757; 4,522,811]. For example, an appropriate ointmentvehicle would contain petrolatum, mineral oil and/or anhydrous liquidlanolin. Sustained release vehicles such as liposomes, membrane orcontact lens delivery systems, or gel-forming plastic polymers wouldalso be suitable delivery vehicles. Liposomes for topical delivery arewell known [see, e.g., U.S. Pat. No. 5,296,231; Mezei et al. (1980)"Liposomes--A selective drug delivery system for the topical route ofadministration, I. lotion dosage form" Life Sciences 26:1473-1477; Mezeiet al. (1981) "Liposomes--A selective drug delivery system for thetopical route of administration: gel dosage form" Journal of Pharmacyand Pharmacology 34:473-474; Gesztes et al. (1988) "Topical anaesthesiaof the skin by liposome-encapsulated tetracaine" Anesthesia andAnalgesia 67:1079-1081; Patel (1985) "Liposomes as a controlled-releasesystem", Biochemical Soc. Trans. 13:513-516; Wohirab et al. (1987)"Penetration kinetics of liposomal hydrocortisone in human skin"Dermatologica 174:18-22].

Liposomes are microcapsules [diameters typically on the order of lessthan 0.1 to 20 μm] that contain selected mixtures and can slowly releasetheir contents in a sustained release fashion. Liposomes or othercapsule, particularly a time release coating, that dissolve uponexposure to oxygen, air, moisture, visible or ultraviolet [UV] light ora particular pH or temperature [see, e., U.S. Pat. No. 4,882,165; Kusumiet al. (1989) Chem. Lett. no.3 433-436; Koch Troels et al. (1990)Bioconjugate Chem. 4:296-304; U.S. Pat. No. 5,482,719; U.S. Pat. No.5,411,730; U.S. Pat. No. 4,891,043; Straubinger et al. (1983) Cell32:1069-1079; and Straubinger et al. (1985) FEBS Lttrs. 179:148-154; andDuzgunes et al. in Chapter 11 of the book CELL FUSION, edited by A. E.Sowers; Ellens et al. (1984) Biochemistry 23:1532-1538; Yatvin et al.(1987) Methods in Enzymology 149:77-87] may be used for example in thesquirt guns or toy machine guns or fairy dust or toy cigarettes.Liposome formulations for use in baking [see, e.g., U.S. Pat. No.4,999,2081 are available. They release their contents when eaten orheated. Such liposomes may be suitable for incorporation into foodproducts herein or in embodiments in which release of the components byheating is desired.

Liposomes be prepared by methods known to those of skill in the art[see, e.g., Kim et al. (1983) Bioch. Bioph. Acta 728:339-348; Assil etal. (1987) Arch Ophthalmol. 105:400; and U.S. Pat. No. 4,522,811, andother citations herein and known to those of skill in the art].

Liposomes that are sensitive to low pH [see, e.g., U.S. Pat. Nos.5,352,448, 5,296,231; 5,283,122; 5,277,913, 4,789,633] are particularlysuitable for addition to bath powders or to bubble compositions, justprior to use. Upon contact with the low pH detergent or soap compositionor a high pH composition, the contents of the liposome will be released.Other components, particularly Ca⁺ or the presence of dissolved O₂ inthe water will cause the components to glow as they are released.Temperature sensitive liposomes are also suitable for use in bathpowders for release into the warm bath water.

c. Encapsulating Vehicles--Gelatin and Polymeric Vehicles

Macro or microcapsules made of gelatin or other such polymer thatdissolve or release their contents in a beverage or food or on contactwith air or light or changes in temperature may also be used toencapsulate components of the bioluminescence generating systems.

Such microcapsules or macrocapsules may also be incorporated into solidsoaps, such that as the soap dissolves the incorporated capsules orpellets release their contents, which glow upon contact with the waterin which the soap is placed.

The aequorin system is particularly suitable for this application. Itcan be encapsulated in suspension or solution or as a paste, or othersuitable form, of buffer with sufficient chelating agent, such as EDTA,to prevent discharge of the bioluminescence. Upon exposure of thecapsule [microcapsule or macrocapsule] to moisture that contains Ca²⁺,such as in a food or beverage, a two chamber apparatus or single chamberapparatus, such as described herein, or even in a moist environmentcontaining Ca²⁺, the slowly released components will glow.

Thus, encapsulated bioluminescence generating components can be used incombination with foods, beverages, ice and ice cubes (and othergeometries of ice), as bullets or pellets, such as "fairy dust" [pelletsthat dissolve upon exposure to light and thereby release theluciferase/luciferin, such as the Renilla system, which will light uponexposure to air], and other such items.

Other encapsulating containers or vehicles for use with thebioluminescence systems are those that dissolve sufficiently in water torelease their contents, or that are readily opened when squeezed in thehand or from which the contents diffuse when mixed with a aqueousmixture. These containers can be made to exclude water, so that thebioluminescence generating system components may be desiccated andplaced therein. Upon exposure to water, such as in an aqueouscomposition or in the atmosphere, the vehicle dissolves or otherwisereleases the contents, and the components react and glow. Similarly,some portion including less than all of the bioluminescence generatingreagents may be provided in pellet form or as a concentrated paste. Forexample, the component(s) may be mixed with gelatin or similar hardeningagent, poured into a mold, if necessary and dried to produce a watersoluble pellet.

The capsules, encapsulating containers or vehicles may be formed fromgelatin or similar water soluble material. If the packaging is to beadded to food or beverage, then it should be chosen to be non-toxic,non-reactive and flavorless. To be readily opened by hand, the packagingmay be constructed of thin plastic or may be configured in two halveswhich form an airtight seal when joined but which are readily separatedwhen release of the components is desired.

In one aspect, these capsular embodiments of the packaging apparatus iscontemplated for use as an additive to beverages, creams, sauces,gelatins or other liquids or semi-solids. In another aspect, it iscontemplated that the contents of the packaging apparatus is releasedinto the air whereby it glows upon contact with the moisture of theatmosphere and/or with molecular oxygen.

d. Endosomes and Vacuoles

Vehicles may be produced using endosomes or vacuoles from recombinanthost cells in which the luciferase is expressed using method known tothose of skill in the art [see, e.g., U.S. Pat. Nos. 5,284,646,5,342,607, 5,352,432, 5,484,589, 5,192,679, 5,206,161, and 5,360,726].For example, aequorin that is produced by expression in a host, such asE. coli, can be isolated within vesicles, such as endosomes or vacuoles,after protein synthesis. Using routine methods the cells are lysed andthe vesicles are released with their contents intact. The vesicles willserve as delivery vehicles. When used they will be charged with aluciferin, such as a coelenterazine, and dissolved oxygen, such as bydiffusion, under pressure, or other appropriate means.

e. Micronized Particles

The bioluminescence generating system components that are suitable forlyophilization, such as the aequorin photoprotein, the Renilla system,and the Vargula systems, can be micronized to form fine powder andstored under desiccating conditions, such as with a desiccant. When usedthe fine powder can be combined with the selected article ofmanufacture, such as a personal item, a chamber in a gun or fountain, orused as fairy dust. Contact with dissolved oxygen or Ca²⁺ in the air orin a mist that can be supplied or in added will cause the particles torelease their contents and glow.

3. Apparatus and Substrates

The combinations herein are produced by combining a selected noveltyitem and combining it with a system and apparatus for producingbioluminescence. Selection of the system depends upon factors such asthe desired color and duration of the bioluminescence desired as well asthe particular item. Selection of the apparatus primarily depends uponthe item with which it is combined.

Among the simplest embodiments herein, are those in which the apparatuscontains a single chamber [vessel] or matrix material and, if needed,ejection means. Components, generally all but at least one necessarycomponent, typically the activator as defined herein, of thebioluminescence reaction are introduced into the housing or vessel oronto the substrate as a mixture in liquid phase or as a powder or otherpaste or other convenient composition. Prior to use the finalcomponent(s) is added or the other components are contacted with thefinal component(s).

a. Matrix Materials

For preparation of combinations of articles of manufacture such asclothing, paper, items fabricated from a textile, plastic, glass,ceramic or other such material, such as a figurine, and for use in thecartridges, at least one component of the bioluminescence generatingsystem is linked to the matrix substrate. When desired, a mixture ormixtures(s) containing the remaining component(s), typically a liquidmixture is applied, as by pouring or spraying onto the matrix substrate,to produce a glow. For example, the aequorin photoprotein, includingcoelenterazine and oxygen, is linked to the substrate. When desired aliquid containing Ca²⁺, such as tap water or, preferably, a liquidmixture containing the Ca²⁺ in an appropriate buffer, is contacted, suchas by spraying, with the matrix with linked luciferase. Upon contactingthe material glows.

In other embodiments, the luciferase, such as a Vargula luciferase, islinked to the substrate material, and contacted with a liquid mixturecontaining the luciferin in an appropriate buffer. Contacting can beeffected by spraying or pouring or other suitable manner. The matrixmaterial is incorporated into, onto or is formed into an article ofmanufacture, such as clothing or a ceramic, glass, plastic figurine,toy, balloon, flocking agent, such as a Christmas tree flocking agent,or other item. The resulting novelty item can be sold as a kit with acontainer of the mixture containing the non-linked components, such asin a canister, spray bottle or can, or other suitable format.

The kits may also include containers containing compositions of thelinked components which can be provided in a form, such as sprayed on asa liquid and air dried, that can be applied to the substrate so that theitem can be made to glow again. Thus, kits containing a substrate, suchas clothing or a plastic, ceramic or glass item, and a first compositioncontaining a luciferase or a luciferin or both and luciferin, and asecond composition containing the remaining components. The item asprovided in the kit can be charged with the first composition, such ashaving the composition applied and dried, or may require charging priorto the first use. Alternatively, the item may be sprayed with bothcompositions when desired to produce a glow.

It is understood that the precise components and optimal means forapplication or storage are a function of the selected bioluminescencesystem. The concentrations of the components, which can be determinedempirically, are not critical, but must be sufficient to produce avisible glow when combined. Typical concentrations are as low asnanomoles/l, preferably on the order of mg/l or higher. Theconcentration on the substrate is that produced when a compositioncontaining such typical concentration is applied to the material. Again,such ideal concentrations can be readily determined empirically byapplying the first composition, letting it dry, spraying the secondcomposition, and observing the result.

The matrix material substrates contemplated herein are generallyinsoluble materials used to immobilize ligands and other molecules, andare those that used in many chemical syntheses and separations. Suchsubstrates, also called matrices, are used, for example, in affinitychromatography, in the immobilization of biologically active materials,and during chemical syntheses of biomolecules, including proteins, aminoacids and other organic molecules and polymers. The preparation of anduse of matrices is well known to those of skill in this art; there aremany such materials and preparations thereof known. For example,naturally-occurring matrix materials, such as agarose and cellulose, maybe isolated from their respective sources, and processed according toknown protocols, and synthetic materials may be prepared in accord withknown protocols.

The substrate matrices are typically insoluble materials that are solid,porous, deformable, or hard, and have any required structure andgeometry, including, but not limited to: beads, pellets, disks,capillaries, hollow fibers, needles, solid fibers, random shapes, thinfilms and membranes. Thus, the item may be fabricated from the matrixmaterial or combined with it, such by coating all or part of the surfaceor impregnating particles.

Typically, when the matrix is particulate, the particles are at leastabout 10-2000 μM, but may be smaller or larger, depending upon theselected application. Selection of the matrices will be governed, atleast in part, by their physical and chemical properties, such assolubility, functional groups, mechanical stability, surface areaswelling propensity, hydrophobic or hydrophilic properties and intendeduse.

If necessary the support matrix material can be treated to contain anappropriate reactive moiety or in some cases the may be obtainedcommercially already containing the reactive moiety, and may therebyserve as the matrix support upon which molecules are linked. Materialscontaining reactive surface moieties such as amino silane linkages,hydroxyl linkages or carboxysilane linkages may be produced by wellestablished surface chemistry techniques involving silanizationreactions, or the like. Examples of these materials are those havingsurface silicon oxide moieties, covalently linked togamma-aminopropylsilane, and other organic moieties;N-[3-(triethyoxysilyl)propyl]phthelamic acid; andbis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readilyavailable materials containing amino group reactive functionalities,include, but are not limited to, para-aminophenyltriethyoxysilane. Alsoderivatized polystyrenes and other such polymers are well known andreadily available to those of skill in this art [e.g., the Tentagel®Resins are available with a multitude of functional groups, and are soldby Rapp Polymere, Tubingen, Germany; see, U.S. Pat. No. 4,908,405 andU.S. Pat. No. 5,292,814; see, also Butz et al. (1994) Peptide Res.7:20-23; Kleine et al. (1994) Immunobiol. 190:53-66].

These matrix materials include any material that can act as a supportmatrix for attachment of the molecules of interest. Such materials areknown to those of skill in this art, and include those that are used asa support matrix. These materials include, but are not limited to,inorganics, natural polymers, and synthetic polymers, including, but arenot limited to: cellulose, cellulose derivatives, acrylic resins, glass,silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers ofvinyl and acrylamide, polystyrene cross-linked with divinylbenzene orthe like [see, Merrifield (1964) Biochemistry 3:1385-1390],polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides,rubber, silicon, plastics, nitrocellulose, celluloses, natural sponges.Of particular interest herein, are highly porous glasses [see, e.g.,U.S. Pat. No. 4,244,721] and others prepared by mixing a borosilicate,alcohol and water.

Synthetic matrices include, but are not limited to: acrylamides,dextran-derivatives and dextran co-polymers, agarose-polyacrylamideblends, other polymers and co-polymers with various functional groups,methacrylate derivatives and co-polymers, polystyrene and polystyrenecopolymers [see, e.g., Merrifield (1964) Biochemistry 3:1385-1390; Berget al. (1990) in Innovation Perspect. Solid Phase Synth. Collect. Pap.,Int. Symp., 1st, Epton, Roger (Ed), pp. 453-459; Berg et al. (1989) inPept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198;Berg et al. (1989) J. Am. Chem. Soc. 111 :8024-8026; Kent et al. (1979)Isr. J. Chem. 17:243-247; Mitchell et al. (1978) J. Org. Chem.43:2845-2852; Mitchell et al. (1976) Tetrahedron Lett. 42:3795-3798;U.S. Pat. No. 4,507,230; U.S. Pat. No. 4,006,117; and U.S. Pat. No.5,389,449]. Methods for preparation of such matrices are well-known tothose of skill in this art.

Synthetic matrices include those made from polymers and co-polymers suchas polyvinylalcohols, acrylates and acrylic acids such aspolyethylene-co-acrylic acid, polyethylene-co-methacrylic acid,polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate,polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid,polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and thosecontaining acid anhydride groups such as polyethylene-co-maleicanhydride, polypropylene-co-maleic anhydride and the like. Liposomeshave also been used as solid supports for affinity purifications [Powerset al. (1989) Biotechnol. Bioeng. 33:173].

For example, U.S. Pat. No. 5,403,750, describes the preparation ofpolyurethane-based polymers. U.S. Pat. No. 4,241,537 describes a plantgrowth medium containing a hydrophilic polyurethane gel compositionprepared from chain-extended polyols; random copolymerization ispreferred with up to 50% propylene oxide units so that the prepolymerwill be a liquid at room temperature. U.S. Pat. No. 3,939,123 describeslightly crosslinked polyurethane polymers of isocyanate terminatedprepolymers containing poly(ethyleneoxy) glycols with up to 35% of apoly(propyleneoxy) glycol or a poly(butyleneoxy) glycol. In producingthese polymers, an organic polyamine is used as a crosslinking agent.Other matrices and preparation thereof are described in U.S. Pat. Nos.4,177,038, 4,175,183, 4,439,585, 4,485,227, 4,569,981, 5,092,992,5,334,640, 5,328,603.

U.S. Pat. No. 4,162,355 describes a polymer suitable for use in affinitychromatography, which is a polymer of an aminimide and a vinyl compoundhaving at least one pendant halo-methyl group. An amine ligand, whichaffords sites for binding in affinity chromatography is coupled to thepolymer by reaction with a portion of the pendant halo-methyl groups andthe remainder of the pendant halo-methyl groups are reacted with anamine containing a pendant hydrophilic group. A method of coating asubstrate with this polymer is also described. An exemplary aminimide is1,1-dimethyl-1-(2-hydroxyoctyl)amine methacrylimide and vinyl compoundis a chloromethyl styrene.

U.S. Pat. No. 4,171,412 describes specific matrices based on hydrophilicpolymeric gels, preferably of a macroporous character, which carrycovalently bonded D-amino acids or peptides that contain D-amino acidunits. The basic support is prepared by copolymerization of hydroxyalkylesters or hydroxyalkylamides of acrylic and methacrylic acid withcrosslinking acrylate or methacrylate comonomers are modified by thereaction with diamines, aminoacids or dicarboxylic acids and theresulting carboxyterminal or aminoterminal groups are condensed withD-analogs of aminoacids or peptides. The peptide containingD-amino-acids also can be synthesized stepwise on the surface of thecarrier.

U.S. Pat. No. 4,178,439 describes a cationic ion exchanger and a methodfor preparation thereof. U.S. Pat. No. 4,180,524 describes chemicalsyntheses on a silica support.

Immobilized Artificial Membranes [IAMs; see, e.g., U.S. Pat. Nos.4,931,498 and 4,927,879] may also be used. IAMs mimic cell membraneenvironments and may be used to bind molecules that preferentiallyassociate with cell membranes [see, e.g., Pidgeon et al. (1990) EnzymeMicrob. Technol. 12:149].

These materials are also used for preparing articles of manufacture,such as toys, balloons, figurines, sponges, knick-knacks, key chains,clothing, translucent or transparent soaps, preferably mild soaps, andother items, and thus are amenable to linkage of molecules, either theluciferase, luciferin, mixtures thereof.

For example, matrix particles may be impregnated into items that willthen be contacted with an activator. For example, matrix particles withlinked luciferin, preferably a luciferin/luciferase complex, such as theaequorin photoprotein is incorporated into a transparent or translucentsoaps [see, e.g., U.S. Pat. Nos. 4,081,394, 5,183,429, and 5,141,664,and United Kingdom Patent No. GB 2,235,931A], preferably a mild soap.Upon contacting the soap with water matrix particles near the surfacewill glow.

Kits containing the item including the matrix material with or withoutthe coating of the bioluminescence generating components, andcompositions containing the remaining components are provided.

b. Immobilization and Activation

Numerous methods have been developed for the immobilization of proteinsand other biomolecules onto solid or liquid supports [see, e.g., Mosbach(1976) Methods in Enzymology 44; Weetall (1975) Immobilized Enzymes,Antigens, Antibodies, and Peptides; and Kennedy et al. (1983) SolidPhase Biochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp.253-391; see, generally, Affinity Techniques. Enzyme Purification: PartB. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby, M. Wilchek, Acad.Press, N.Y. (1974); Immobilized Biochemicals and AffinityChromatography, Advances in Experimental Medicine and Biology, vol. 42,ed. R. Dunlap, Plenum Press, N.Y. (1974)].

Among the most commonly used methods are absorption and adsorption orcovalent binding to the support, either directly or via a linker, suchas the numerous disulfide linkages, thioether bonds, hindered disulfidebonds, and covalent bonds between free reactive groups, such as amineand thiol groups, known to those of skill in art [see, e.g., the PIERCECATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, which describesthe preparation of and use of such reagents and provides a commercialsource for such reagents; and Wong (1993) Chemistry of ProteinConjugation and Cross Linking, CRC Press; see, also DeWitt et al. (1993)Proc. Natl. Acad. Sci. U.S.A. 90:6909; Zuckermann et al. (1992) J. Am.Chem. Soc. 114:10646; Chem et al. (1994) J. Am. Chem. Soc. 116:2661;Bunnin et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:4708; Sucholeiki(1994) Tetrahedron Lttrs. 35:7307; and Su-Sun Wang (1976) J. Org. Chem.41:3258; Padwa et al. (1971) J. Org. Chem. 41:3550 and Vedejs et al.(1984) J. Org. Chem. 49:575, which describe photosensitive linkers].

To effect immobilization, a composition containing the protein or otherbiomolecule is contacted with a support material such as alumina,carbon, an ion-exchange resin, cellulose, glass or a ceramic.Fluorocarbon polymers have been used as supports to which biomoleculeshave been attached by adsorption [see, U.S. Pat. No. 3,843,443;Published International PCT Application WO/86 03840].

A large variety of methods are known for attaching biological molecules,including proteins and nucleic acids, molecules to solid supports [see.e.g., U.S. Pat. No. 5451683]. For example, U.S. Pat. No. 4,681,870describes a method for introducing free amino or carboxyl groups onto asilica matrix. These groups may subsequently be covalently linked toother groups, such as a protein or other anti-ligand, in the presence ofa carbodiimide. Alternatively, a silica matrix may be activated bytreatment with a cyanogen halide under alkaline conditions. Theanti-ligand is covalently attached to the surface upon addition to theactivated surface. Another method involves modification of a polymersurface through the successive application of multiple layers of biotin,avidin and extenders [see, e.g., U.S. Pat. No. 4,282,287]; other methodsinvolve photoactivation in which a polypeptide chain is attached to asolid substrate by incorporating a light-sensitive unnatural amino acidgroup into the polypeptide chain and exposing the product to low-energyultraviolet light [see, e.g., U.S. Pat. No. 4,762,881]. Oligonucleotideshave also been attached using a photochemically active reagents, such asa psoralen compound, and a coupling agent, which attaches thephotoreagent to the substrate [see, e.g., U.S. Pat. No. 4,542,102 andU.S. Pat. No. 4,562,157]. Photoactivation of the photoreagent binds anucleic acid molecule to the substrate to give a surface-bound probe.

Covalent binding of the protein or other biomolecule or organic moleculeor biological particle to chemically activated solid matrix supportssuch as glass, synthetic polymers, and cross-linked polysaccharides is amore frequently used immobilization technique. The molecule orbiological particle may be directly linked to the matrix support orlinked via linker, such as a metal [see, e.g., U.S. Pat. No. 4,179,402;and Smith et al. (1992) Methods: A Companion to Methods in Enz.4:73-78]. An example of this method is the cyanogen bromide activationof polysaccharide supports, such as agarose. The use of perfluorocarbonpolymer-based supports for enzyme immobilization and affinitychromatography is described in U.S. Pat. No. 4,885,250]. In this methodthe biomolecule is first modified by reaction with a perfluoroalkylatingagent such as perfluorooctylpropylisocyanate described in U.S. Pat. No.4,954,444. Then, the modified protein is adsorbed onto the fluorocarbonsupport to effect immobilization.

The activation and use of matrices are well known and may be effected byany such known methods [see, eg., Hermanson et al. (1992) ImmobilizedAffinity Ligand Techniques, Academic Press, Inc., San Diego]. Forexample, the coupling of the amino acids may be accomplished bytechniques familiar to those in the art and provided, for example, inStewart and Young, 1984, Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford.

Other suitable methods for linking molecules to solid supports are wellknown to those of skill in this art [see, e.g., U.S. Pat. No.5,416,193]. These include linkers that are suitable for chemicallylinking molecules, such as proteins, to supports and include, but arenot limited to, disulfide bonds, thioether bonds, hindered disulfidebonds, and covalent bonds between free reactive groups, such as amineand thiol groups. These bonds can be produced using heterobifunctionalreagents to produce reactive thiol groups on one or both of the moietiesand then reacting the thiol groups on one moiety with reactive thiolgroups or amine groups to which reactive maleimido groups or thiolgroups can be attached on the other. Other linkers include, acidcleavable linkers, such as bismaleimideothoxy propane, acidlabile-transferrin conjugates and adipic acid diihydrazide, that wouldbe cleaved in more acidic intracellular compartments; cross linkers thatare cleaved upon exposure to UV or visible light and linkers, such asthe various domains, such as C_(H) 1, C_(H) 2, and C_(H) 3, from theconstant region of human IgG₁ (see, Batra et al. (1993) MolecularImmunol. 30:379-386). Presently preferred linkages are direct linkageseffected by adsorbing the molecule to the surface of the matrix. Otherlinkages are photocleavable linkages that can be activated by exposureto light [see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107,which linkers are herein incorporated by reference]. The photocleavablelinker is selected such that the cleaving wavelength that does notdamage linked moieties. Photocleavable linkers are linkers that arecleaved upon exposure to light [see, e.g., Hazum et al. (1981) in Pept.,Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp. 105-110, whichdescribes the use of a nitrobenzyl group as a photocleavable protectivegroup for cysteine; Yen et al. (1989) Makromol. Chem 190:69-82, whichdescribes water soluble photocleavable copolymers, includinghydroxypropylmethacrylamide copolymer, glycine copolymer, fluoresceincopolymer and methylrhodamine copolymer; Goldmacher et al. (1992)Bioconj. Chem. 3:104-107, which describes a cross-linker and reagentthat undergoes photolytic degradation upon exposure to near UV light(350 nm); and Senter et al. (1985) Photochem. Photobiol 42:231-237,which describes nitrobenzyloxycarbonyl chloride cross linking reagentsthat produce photocleavable linkages]. The selected linker will dependupon the particular application and, if needed, may be empiricallyselected.

Aequorin that is designed for conjugation and conjugates containing suchaequorin have been produced [see, e.g., International PCT applicationNo. WO 94/18342; see, also Smith et al. (1995) in American BiotechnologyLaboratory]. Vargula luciferase has also been linked to other molecules[see, e.g., Japanese application No. JP 5064583, Mar. 19, 1993]. Suchmethods may be adapted for use herein to produce aequorin coupled toprotein or other such molecules, which are linked to the selectedmatrix. Finally, as an alternative, a component of the bioluminescencegenerating system may be modified for linkage, such as by addition ofamino acid residues that are particularly suitable for linkage to theselected substrate. This can be readily effected by modifying the DNAand expressing such modified DNA to produce luciferase with additionalresidues at the N- or C-terminus.

4. Apparatus Containing a Single Chamber, Housing or a Vessel

Examples of vessels include beverage containers, plates or other dishes,vases, jars, balloons, bottles and other containers.

Single chamber housings or vessels will include single chamber waterguns, inks, paints and other such items, in which one or more componentsof the bioluminescence system up to all of the components except for oneof the components required for bioluminescence is included in the vesselas a mixture, powder or suspension of particles. The remainingcomponent(s) is(are) introduced just prior to use. Thus, for example,for a squirt gun or a balloon or other such item, the items can bepackaged with a powder in the chamber or inside the item, or a powder orother composition can be added, and then water is added. Alternatively,the luciferase, such as Renilla, Vargula, and firefly luciferase, can belinked to the surface of the item and water added. Depending upon thebioluminescence generating system selected the water can be tap water orwater that contains the additional component, such as dissolved oxygen,or Ca²⁺ or ATP, or other suitable composition, and/or appropriateluciferin/bioluminescence substrate. Similarly, the luciferase/luciferase can be linked to the surface of the item in association withthe appropriate luciferin/bioluminescence substrate, such that additionof activator alone generates luminescence.

For inks or paints the components are suspended in the ink or paint, andthen the final component(s) is(are) added. Alternatively, pelletscontaining components of the bioluminescence generating system, such asthe Renilla or Aequorin system can be added to an ink or paint or othersuch liquid item, and as the pellet dissolves or the contents diffuseout, the item will glow.

Kits containing the item and the bioluminescence generating systems arealso provided herein. The kits typically contain a beverage container,balloon or bottle and, may also contain, the buffer compositions andother ingredients required for the bioluminescence reaction, as well asinstructions for use. The kits may also include the cartridges forrecharging or reloading the item.

5. Dual and Multiple Chamber Fluid Dispensing Apparatus

An example of a dispensing apparatus contemplated for use herein is adual chamber fluid dispensing apparatus. In general, this apparatus hastwo chambers thereby maintaining at least one of the bioluminescencegenerating system components separate from the remaining componentsuntil illumination is desired. This apparatus may include a mixingchamber to permit mixing of the components prior to dispensing from theapparatus. Further, the apparatus may be used with fluid or semi-fluidbioluminescence systems; for example, water based compositions orcream/lotion systems.

a. Mechanical Pump Dispensing Apparatus

Another embodiment of a dual chamber fluid dispensing apparatus employsa mechanical pump mechanism in its operation. In this embodiment, thedispensing apparatus maintains at least one of the components of thebioluminescence reaction, such as the substrate, luciferase oractivator, in separate chambers. A pump mechanism operates to withdrawthe contents from each chamber and into a mixing chamber. Within themixing chamber and upon ejection, the mixed composition is activated,for example by the oxygen in the air or by reaction of the componentsthat were in one chamber, and glows. The pump mechanism may be manuallyoperated, for example by pulling the trigger of a toy squirt gun, or itmay be mechanically operated, for example by a motor which operates thepumping mechanism.

b. Gas-Charged Dispensing Apparatus

Another example of a dual chamber fluid dispensing apparatus is one thatuses CO₂ or, preferably a mixture gases containing O₂, or other gas, topropel the components of the bioluminescence system, such as thebioluminescence substrate and luciferase into a mixing chamber wherethey combine before being ejected through a dispensing nozzle. In such adispensing apparatus, upon mixing of the contents in the mixing chamberthe contents will glow.

These apparatus may be configured as, for example, a toy gun, toy cannonor other toy weapon, a can for shaving cream or other glowing foam, adecorative fountain or volcano or almost any fluid squirting or spoutingdevice. A volcano shaped dispensing apparatus may be used, for example,as a substitute for conventional, similarly shaped fireworks displays.

Almost any bioluminescence generating system may be selected for usewith the dual chamber fluid dispensing apparatus. If air is thebioluminescence activator, then the contents glow after mixing and uponejection from the dispensing apparatus. Alternatively, thebioluminescent activator may be contained in one of the two chambersalong with either the luciferase or bioluminescence substrate, or it maybe located in a third chamber that is also connected to the mixingchamber. Thus, as with all the combinations described herein, thecritical aspect of these dispensing apparatus is that at least one ofthe bioluminescence generating system components be maintained separatefrom the other components until reaction is desired.

c. Compressible Dispensing apparatus

Another embodiment of a dual chamber fluid dispensing apparatuscontemplated for use herein takes the form of a compressible bottle ortube. The bottle has two compartments within it that keep at least twoof the bioluminescence generating system components separated. The capof the bottle can serve as a mixing chamber or a mixing chamber may bepositioned between the two chambers and the cap. The bioluminescencegenerating system components are forced by compression from the bottleinto the mixing chamber. They are then dispensed from the mixingchamber. For example, the mixed contents may be removed from the bottleby attaching a plunger/syringe apparatus to the dispensing end andwithdrawing the contents therethrough.

Such compressible bottle or tube is particularly useful for dispensingbioluminescent body creams, gels or lotions, finger paints, dentifrices,shampoos, hair gels, cosmetics and other viscous fluids and semi-solids.The bottle or tube is preferably constructed of plastic, plastic/metallaminate or similar collapsible composite to avoid formation of a vacuumwithin the container as its contents are expelled. See, for example,U.S. Pat. No. 4,687,663, which describes a dual chambered tube for usewith dentifrices and which, as all cited patents and publicationsherein, is incorporated herein in its entirety. This tube may be adaptedfor use in combination with the bioluminescence generating systemsprovided herein. Other tubes and vessels that have dual chambers, suchas those used to keep components of the final product separate untiluse, may be used herein [see, e.q., U.S. Pat. Nos. 5,405,056, 4,676,406,4,438,869, 5,059,417, 4,528,180, 4,849,213, 4,895,721, 5,085,853, see,esp. 5,038,963]

6. Other fluid dispensing and packaging apparatus particularly designedfor single use

Additional embodiments of the dispensing and packaging apparatuscontemplated for use herein include fluid packaging apparatus, designedfor use with bioluminescent fluids. These apparatus maintain at leastone of the bioluminescence generating system components separate fromthe remaining components until illumination is desired. Unlike the dualchamber fluid dispensing apparatus, however, these apparatus result inillumination of the entire contents of the package and therefore aretypically intended for a single use applications. They can, however, berecharged by adding additional substrate, luciferase or other exhaustedcomponent.

a. Bottle-type single chamber container/bladder apparatus

One example of a fluid packaging apparatus, contemplated for use herein,is a bottle shaped device having a bladder within it that contains atleast one of the bioluminescence generating system components. Apiercing pin or other means for rupturing the bladder is also locatedwithin the bottle. When the bladder is ruptured, within the bottle, itscontents mix with the contents of the bottle and the resulting mixturebecomes illuminated or glows upon contact with an activator, such asair.

Because the bioluminescence generating system components are mixedwithin the entire bottle, those contents must be used shortly aftermixing. Thus, this type of packaging is particularly suitable for usewith bioluminescence systems that are consumed in a single use oractivity such as bubble-blowing.

b. Dual chambered bottle type container/bladder apparatus for use withfoods and beverages

Another example of a fluid packaging apparatus provided herein is asingle use, dual chambered bottle. This apparatus is configured with amembrane between the two chambers. One chamber is designed to readilycollapse against the other chamber thereby rupturing the membrane whichdivides the chambers. The contents of the two chambers then mix,resulting in illumination of the fluids. Alternatively, instead of amembrane separation means, a one-way valve may be situated between thetwo chambers. Such a single use, dual chamber apparatus is particularlysuitable for use with bubble-making compositions, beverages, single useamounts of shampoos, soaps, creams or lotions, or similar substances.

c. Can type container/bladder apparatus for use with foods and beverages

Another example of a fluid packaging apparatus, which is amenable to usewith bioluminescent food or beverage, is a container/bladdercombination. In one embodiment, the container is configured like apop-top can, such as a soda can. A bladder, containing at least one ofthe bioluminescence generating system components, is positioned underthe top of the can. Within the can is a beverage that contains theremaining bioluminescence generating system components. Upon opening thecan, the bladder is punctured and its contents mixed with the rest ofthe contents of the can; thereby illuminating the beverage. Preferably,the container is clear, so that the illumination will be almostimmediately visible. Other pop top cans that can be modified for useherein are known [see, e.g., U.S. Pat. No. 5,397,014].

Alternative configurations of the container/bladder apparatus arelikewise contemplated. For example, the container may be in any shapeand configured with a removable cap to which the bladder is attached. Tocause the beverage to glow, the bladder is punctured or otherwisecompromised and its contents added to the container; thereby causingillumination of the food or beverage. The contents of the container neednot be a food or beverage, any fluid or semi-solid may be used and isherein contemplated.

d. Spray containers that produce a glowing spray

Spray containers or cans that are adapted to produce a glowing spray areprovided herein. These containers are also intended for use in anyapplication in which two components, particularly solutions or liquidcomponents, are intended to be mixed just prior to use. These containersinclude a housing portion for the first component and a second portiondesigned to inject or introduce the second component.

A preferred embodiment of these containers, which is illustrated inFIGS. 20-22 [see, also EXAMPLE 10], includes two portions, a top housingportion and a bottom plunger portion. For use in generatingbioluminescence, the top housing portion includes all, except one ormore, of the components of a bioluminescence generating system. Theremaining components of the bioluminescence generating system arecontained in a pellet or are encapsulated, as described above.

The top housing portion is adapted at its bottom end with an indentationwithin which the pellet fits. At least one wall of the indentationincludes a rupturable membrane or material. The top housing portion isfurther adapted to attach securely to and within the bottom plungerportion. A plunger is situated within the bottom plunger portion suchthat the plunger rests in the indentation of the top housing portionwhen the bottom plunger portion is tightly secured thereto. Inoperation, the pellet or encapsulated vehicle is placed within theindentation of the top housing portion and the bottom plunger portionsecured tightly thereto. The plunger within the bottom plunger portionpresses against the pellet forcing it through the rupturable membrane ormaterial, thereby permitting the pellet to dissolve in and mix with thecontents of the top housing portion. Alternatively the pellet willinclude a sharpened portion that will puncture the rupturable wall ofthe housing. An angular seal may be used, situated within the bottomplunger portion, to set against the bottom of the top housing portionforming a seal to prevent leakage of the mixed contents of the spray canapparatus. The top housing portion additionally contains a conduit orother suitable means for ejecting the contents.

The top housing portion of the spray container may be adapted to receivethe bottom plunger portion by threading the two spray can portions sothat they may be screwed together. [See, e.g., FIG. 21, illustrating thespray container apparatus with the bottom plunger portion fully screwedinto place]. Alternatively, the two portions may be adapted to snaptogether, such as by insertion of a tongue from one portion into agroove of the other portion.

As stated, the indentation or pocket located in the bottom end of thetop housing portion includes at least one wall formed by a rupturablemembrane. Preferably that wall is the top wall and is readily rupturedby pressure, for example, from the pellet or plunger or plunger forcingthe pellet, against it. The pellet is fabricated from material that willrelease the contents into aqueous medium. The pellet may also include asharp tip designed to puncture the spray container.

The spray container is fabricated from suitable materials, such asplastic, aluminum, metal alloys, tin, and other materials from whichspray cans and containers, such as hair spray cans and other containersdesigned for delivery of aerosols and sprays, are fabricated. The sizeof the spray can apparatus may vary depending upon the intended use anddemands of the market place, but will typically have a usable volume offrom about 100 mls to about a liter.

The bottom plunger portion is typically fabricated from a metal, such asaluminum, and the plunger is shaped and situated such that it fits intothe pocket of the top housing portion when the bottom plunger portion isscrewed tightly in place. It can also be made from compressible plasticor other such material and designed to compress and deliver the insertedpellet, which is designed to fit into the indentation, slot or pocketand be retained by virtue of the tight fit.

7. Cap Apparatus for use a single chamber vessel

Another example of a packaging apparatus contemplated herein is a capapparatus for use with a vessel. In this embodiment, one or more of thebioluminescence generating system components, up to all but onecomponent, is [are] within the cap of the vessel and the remainingcomponents are contained in the vessel. Upon operation of the capapparatus, the bioluminescence generating system components are added tothe composition in the vessel and the composition glows. Preferably thevessel is translucent to the bioluminescence; however, the glowingcomposition may be dispensed from the vessel.

Generally, the cap is configured with a pocket within it which opens tothe bottom of the cap. For example, the bottom of the cap can beU-shaped, curving into the cap and thereby forming the pocket. The capapparatus contains a capsule or similar package, containing one or more,up to all but one, of the bioluminescence generating system components,within the pocket in the cap. Means for deploying the bioluminescencegenerating system components into the vessel are attached to the cap.Such deployment means can be, for example, a plunger assembly. The capapparatus is operated by depressing the plunger, thereby forcing thepackaged components into the composition within the vessel or breakingthe packaging, releasing its contents into the composition within thevessel. The package should be dissolvable in the composition or amenableto diffusion of the components contained therein or readily rupturableupon contact with the plunger assembly.

Alternatively, the packaging within the cap apparatus can be a membraneor series of membranes separating the bioluminescence generating systemcomponents from the composition within the vessel or from thecomposition within the vessel and from each other. In this alternative,the plunger can rupture the membrane(s) thereby permitting thebioluminescence generating system components contained therein to bereleased into the composition contained in the vessel. Again, uponmixture of the components with the composition, illumination ensues.

The bioluminescence generating system components contained within thecap apparatus may be in a composition, such as a solution, a powder or asuspension of particles or other form amenable to packaging within thecap apparatus that can be mixed with the composition contained withinthe vessel. The cap apparatus also may be adapted with a screen orfilter attached to the bottom of the cap to prevent membrane fragmentsfrom entering the vessel.

The cap apparatus, as all the apparatus described herein that are incontact with a bioluminescence generating system component, should benon-reactive with the components and is preferably non-toxic,particularly if used with a composition intended for human consumption.The cap can be constructed of cork, for example, and situated in a wineor champagne bottle. Alternatively, the cap can be a screw-top type cap,having a plunger integral thereto, such that tightening of the screw-caponto the top of the vessel forces the plunger against the packagedbioluminescence generating system components either rupturing thepackaging or pushing it into the vessel.

E. Combinations of articles of manufacture and bioluminescence

Combinations of articles of manufacture and bioluminescence are providedherein. By virtue of the bioluminescence the combinations are noveltyitems because the bioluminescence provides entertainment, amusement orrecreation. Any such combination of an article of manufacture withbioluminescence that produces a novelty item [i.e., providesentertainment, amusement, or recreation] is intended herein. Thecombination is formed by contacting the article of manufacture ormaterials in the manufacture with a bioluminescence generating system oran apparatus therefore. The components of the bioluminescence generatingsystem are manufactured as part of the item, coated thereon, impregnatedtherein, or added after manufacture. Alternatively, the article ofmanufacture is combined with an apparatus that contains or to whichcomponents of the bioluminescence generating system are added, and thatproduces the bioluminescence.

The bioluminescence generating systems provided herein are contemplatedfor use with various substances to glow the substance. For example, asdiscussed below, the bioluminescence generating system components may beused to produce glowing aqueous mixtures housed in transparent portionsof articles of manufacture, thereby illuminating that portion of thearticle of manufacture. Additionally, the bioluminescence generatingsystem components may be used to produce glowing food or beverageproducts, textiles, creams, lotions, gels, soaps, bubbles, papers,powders or water. Following are brief examples of combinations ofbioluminescence systems with articles of manufacture and the resultingnovelty items contemplated herein.

1. Personal care products, including bath powders, bubble baths,products for use on the nails, hair, skin, lips and elsewhere

Personal care products can be in the form of powders, pressed powders,sprays, foams, aerosols, lotions, gels, ointments and other suitableformulations. The common element will be the combination of such itemswith bioluminescence generating reagents or fluorescent proteins, sothat before use or upon application to the body or when used the productwill glow. These items include, body powders, lotions, gels, aqueouscompositions and solutions, nail polishes, make-up, body paints, shavingcream and dentifrices. As described herein, the items are combined withone or more components of a bioluminescence generating system, and, whena glow is desired, the remaining components are added or combined withthe other components.

a. Bath powders

Numerous bath powders exemplified herein, are suitable for use incombination with the bioluminescence generating systems herein. Suchbath powders are preferably non-detergent with a pH close to neutral.The selected bioluminescence generating system must be selected to beactive at the resulting pH. In addition, capsular delivery vehicles,such as liposomes or time release delivery vehicles, preferablymicrocapsules, that contain a luciferase and luciferin, such as theRenilla, Vargula, or Aequorin system, and that are pH, temperaturesensitive, or that dissolve in water or that are otherwise released arepreferred for use herein. In certain embodiments, there will be twotypes of capsules, one type containing up to all but one of thecomponents required for the bioluminescence reaction, and the othercontaining the remaining components [except, if desired, for thosecomponents that will be present in the bath water, such as Ca²⁺ ]. Suchcapsules may be components of the bath powder or may be added to a bathto give it a glow. Upon contact with the warm water or with water of aparticular pH the contents of the capsule or pellet will be released,preferably over time, and will glow.

In other embodiments, there will be one type of capsule that containsthe luciferase and other components. The luciferin may be included inthe bath powder or added separately. Other ways in which the componentsmay be combined will, in light of the disclosure herein, be apparent tothose of skill in the art. The bath powders and bioluminescencegenerating reactions will be provided as a combination or in a kit.

Suitable bath powders and bubble baths and other bubble compositions foruse in these combinations are well known to those of skill in the art[see, e.g., U.S. Pat. Nos.: 5,478,501 4,565,647; 5,478,490; 5,412,118;5,401,773; and many other examples]. These may be modified by adding thebioluminescence generating system components.

b. Glowing dust or powder

Another embodiment of the combination described herein is as a glowingdust or powder substance, or a vapor, such as for use in the theatricalproductions. In this embodiment, lyophilized or desiccated forms,micronized powdered forms, or, a suitable composition, of up to all butone of the bioluminescence generating system components are encapsulatedin readily rupturable or time release or temperature or pH or lightsensitive microspheres or capsules, as described above. Preferableencapsulating agents are light or temperature sensitive so that uponexposure to the environment, the contents are released from thecapsules. Moisture or oxygen in the air or a spray of water on the skinwith dissolved oxygen in the vicinity of the "dust" will produce a glow.

The dust can be added to another powder, such as body powder, providedit is stored in an airtight container. Once the powder contacts themoisture in the air and on the wearer's skin, it glows.

Alternatively, micronized particles of lyophilized powders are packagedsuch in manner so that the powder remains dry. Upon exposure to moistair or to air with water droplets [such as a fog], the micronizedpowders will glow.

c. Lotions, gels and other topical application formulations

For application to the skin, the macro or microparticles or theluciferase, luciferin or mixture thereof, may be added to cosmeticcompositions. The compositions may be provided in the form of gels,creams, lotions, solids, and other compositions, such as solutions andsuspensions, aerosols or solid base or vehicle known in the art to benon-toxic and dermatologically acceptable to which sufficient number ofsuch particles are added under conditions in which the contents arereleased into the gels, creams, lotions, solids, solutions orsuspensions, or aerosols, which contain either molecular oxygen and/orCa²⁺ to react with the contents of particles. Upon application to theskin the gels, creams, lotions, solids, solutions or suspensions, oraerosols glow.

(1) Lotions

The lotions contain an effective concentration of less than all reagentsfor one or more bioluminescence generating systems. Preferably, thereagents are encapsulated in a vehicle that releases its contents uponexposure to light or temperature, such that as the contents of thevehicle are released they react with oxygen or Ca²⁺ in the lotion and/oron the skin. Prior to use the skin can be sprayed with a mist of water,buffer or other composition containing the requisite ions. The effectiveconcentration is that sufficient to produce a visible glow whencontacting the skin. Any emollients, as long as they do not inactivatethe bioluminescent reaction, known to those of skill in the art assuitable for application to human skin may be used. These include, butare not limited to, the following:

(a) Hydrocarbon oils and waxes, including mineral oil, petrolatum,paraffin, ceresin, ozokerite, microcrystalline wax, polyethylene, andperhydrosqualene.

(b) Silicone oils, including dimethylpolysiloxanes,methylphenylpolysiloxanes, water-soluble and alcohol-solublesilicone-glycol copolymers.

(c) Triglyceride fats and oils, including those derived from vegetable,animal and marine sources. Examples include. but are not limited to,castor oil, safflower oil, cotton seed oil, corn oil, olive oil, codliver oil, almond oil, avocado oil, palm oil, sesame oil, and soybeanoil.

(d) Acetoglyceride esters, such as acetylated monoglycerides.

(e) Ethoxylated glycerides, such as ethoxylated glyceryl monstearate.

(f) Alkyl esters of fatty acids having 10 to 20 carbon atoms. Methyl,isopropyl and butyl esters of fatty acids are useful herein. Examplesinclude, but are not limited to, hexyl laurate, isohexyl laurate,isohexyl palmitate, isopropyl palmitate, isopropyl myristate, decyloleate, isodecyl oleate, hexadecyl stearate, decyl stearate, isopropylisostearate, diisopropyl adipate, diisohexyl adipate, dihexyidecyladipate, diisopropyl sebacate, lauryl lactate, myristyl lactate, andcetyl lactate.

(g) Alkenyl esters of fatty acids having 10 to 20 carbon atoms. Examplesthereof include, but are not limited to, oleyl myristate, oleylstearate, and oleyl oleate.

(h) Fatty acids having 9 to 22 carbon atoms. Suitable examples include,but are not limited to, pelargonic, lauric, myristic, palmitic, stearic,isostearic, hydroxystearic, oleic, linoleic, ricinoleic, arachidonic,behenic, and erucic acids.

(i) Fatty alcohols having 10 to 22 carbon atoms, such as, but notlimited to, lauryl, myristyl, cetyl, hexadecyl, stearyl, isostearyl,hydroxystearyl, oleyl, ricinoleyl, behenyl, erucyl, and 2-octyl dodecylalcohols.

(j) Fatty alcohol ethers, including, but not limited to ethoxylatedfatty alcohols of 10 to 20 carbon atoms, such as, but are not limitedto, the lauryl, cetyl, stearyl, isostearyl, oleyl, and cholesterolalcohols having attached thereto from 1 to 50 ethylene oxide groups or 1to 50 propylene oxide groups or mixtures thereof.

(k) Ether-esters, such as fatty acid esters of ethoxylated fattyalcohols.

(l) Lanolin and derivatives, including, but not limited to, lanolin,lanolin oil, lanolin wax, lanolin alcohols, lanolin fatty acids,isopropyl lanolate, ethoxylated lanolin, ethoxylated lanolin alcohols,ethoxylated cholesterol, propoxylated lanolin alcohols, acetylatedlanolin, acetylated lanolin alcohols, lanolin alcohols linoleate,lanolin alcohols ricinoleate, acetate of lanolin alcohols ricinoleate,acetate of ethoxylated alcohols-esters, hydrogenolysis of lanolin,ethoxylated hydrogenated lanolin, ethoxylated sorbitol lanolin, andliquid and semisolid lanolin absorption bases.

(m) Polyhydric alcohols and polyether derivatives, including, but notlimited to, propylene glycol, dipropylene glycol, polypropylene glycol[M.W. 2000-4000], polyoxyethylene polyoxy-propylene glycols,polyoxypropylene polyoxyethylene glycols, glycerol, ethoxylatedglycerol, propoxylated glycerol, sorbitol, ethoxylated sorbitol,hydroxypropyl sorbitol, polyethylene glycol [M.W. 200-6000], methoxypolyethylene glycols 350, 550, 750, 2000, 5000, poly(ethylene oxide)homopolymers [M.W. 100,000-5,000,000], polyalkylene glycols andderivatives, hexylene glycol (2-methyl-2,4-pentanediol), 1,3-butyleneglycol, 1,2,6,-hexanetriol, ethohexadiol USP (2-ethyl-1,3-hexanediol),C₁₅ -C₁₈ vicinal glycol and polyoxypropylene derivatives oftrimethylolpropane.

(n) Polyhydric alcohol esters, including, but not limited to, ethyleneglycol mono- and di-fatty acid esters, diethylene glycol mono- anddi-fatty acid esters, polyethylene glycol [M.W. 200-6000], mono- anddi-fatty esters, propylene glycol mono- and di-fatty acid esters,polypropylene glycol 2000 monooleate, polypropylene glycol 2000monostearate, ethoxylated propylene glycol monostearate, glyceryl mono-and di-fatty acid esters, polyglycerol poly-fatty acid esters,ethoxylated glyceryl monostearate, 1,3-butylene glycol monostearate,1,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester,sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acidesters.

(o) Wax esters, including, but not limited to, beeswax, spermaceti,myristyl myristate, and stearyl stearate and beeswax derivatives,including, but not limited to, polyoxyethylene sorbitol beeswax, whichare reaction products of beeswax with ethoxylated sorbitol of varyingethylene oxide content that form a mixture of ether-esters.

(p) Vegetable waxes, including, but not limited to, carnauba andcandelilla waxes.

(q) Phospholipids, such as lecithin and derivatives.

(r) Sterols, including, but not limited to, cholesterol and cholesterolfatty acid esters.

(s) Amides, such as fatty acid amides, ceramides, ethoxylated fatty acidamides, and solid fatty acid alkanolamides.

The lotions further preferably contain [by weight] from 1% to 10%, morepreferably from 2% to 5%, of an emulsifier. The emulsifiers can benonionic, anionic or cationic. Examples of satisfactory nonionicemulsifiers include, but are not limited to, fatty alcohols having 10 to20 carbon atoms, fatty alcohols having 10 to 20 carbon atoms condensedwith 2 to 20 moles of ethylene oxide or propylene oxide, alkyl phenolswith 6 to 12 carbon atoms in the alkyl chain condensed with 2 to 20moles of ethylene oxide, mono- and di-fatty acid esters of ethyleneoxide, mono- and di-fatty acid esters of ethylene glycol where the fattyacid moiety contains from 10 to 20 carbon atoms, diethylene glycol,polyethylene glycols of molecular weight 200 to 6000, propylene glycolsof molecular weight 200 to 3000, glycerol, sorbitol, sorbitan,polyoxyethylene sorbitol, polyoxyethylene sorbitan and hydrophilic waxesters. Suitable anionic emulsifiers include, but are not limited to,the fatty acid soaps, e.g. sodium, potassium and triethanolamine soaps,where the fatty acid moiety contains from 10 to 20 carbon atoms. Othersuitable anionic emulsifiers include, but are not limited to, the alkalimetal, ammonium or substituted ammonium alkyl sulfates, alkylarylsulfonates, and alkyl ethoxy ether sulfonates having 10 to 30 carbonatoms in the alkyl moiety. The alkyl ethoxy ether sulfonates containfrom 1 to 50 ethylene oxide units. Among satisfactory cationicemulsifiers are quaternary ammonium, morpholinium and pyridiniumcompounds. Certain of the emollients described in preceding paragraphsalso have emulsifying properties. When a lotion is formulated containingsuch an emollient, an additional emulsifier is not needed, though it canbe included in the composition.

Other conventional components of such lotions may be included. One suchadditive is a thickening agent at a level from 1% to 10% by weight ofthe composition. Examples of suitable thickening agents include, but arenot limited to: cross-linked carboxypolymethylene polymers, ethylcellulose, polyethylene glycols, gum tragacanth, gum kharaya, xanthangums and bentonite, hydroxyethyl cellulose, and hydroxypropyl cellulose.

The balance of the lotion is water or a C₂ or C₃ alcohol, or a mixtureof water and the alcohol. The lotions are formulated by admixing all ofthe components together. Preferably bioluminescence generating systemreagents are suspended or otherwise uniformly dispersed in the mixture.

In certain embodiments the components may be mixed just prior to use.Devices for effecting such mixture are known to those of skill in theart or are exemplified herein.

Kits containing the lotion and powders, capsular vehicles and,optionally, buffer compositions containing ATP, Ca²⁺ and otheringredients required for the bioluminescence reaction are also provided.

(2) Creams

The creams are similarly formulated to contain an effectiveconcentration typically at between about 0.1%, preferably at greaterthan 1% up to and greater than 50%, preferably between about 3% and 50%,more preferably between about 5% and 15% [by weight] of one ore more thebioluminescence generating systems provided herein. The creams alsocontain from 5% to 50%, preferably from 10% to 25%, of an emollient andthe remainder is water or other suitable non-toxic carrier, such as anisotonic buffer. The emollients, as described above for the lotions, canalso be used in the cream compositions. The cream may also contain asuitable emulsifier, as described above. The emulsifier is included isin the composition at a level from 3% to 50%, preferably from 5% to 20%.

(3) Solutions and suspensions for topical application

These compositions are formulated to contain an amount sufficient toproduce a visible glow, typically at a concentration of between about0.1-10 mg/l preferably between 1 and 5 mg/l of the luciferase. Theamount of luciferin is similarly between about 0.1 and 10 mg/l, althoughthe amount can be selected based on the desired duration of the glow.The balance is water, a suitable organic solvent or other suitablesolvent or buffer. Suitable organic materials useful as the solvent or apart of a solvent system are as follows: propylene glycol, polyethyleneglycol [M.W. 200-600], polypropylene glycol [M.W. 425-2025], glycerine,sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, diethyltartrate, butanediol, and mixtures thereof. Such solvent systems canalso contain water.

Solutions or suspensions used for topical application can include any ofthe following components: a diluent, such as water saline solution,fixed oil, polyethylene glycol, glycerine, propylene glycol or othersynthetic solvent; antimicrobial agents, such as benzyl alcohol andmethyl parabens; antioxidants, such as ascorbic acid and sodiumbisulfite; chelating agents, such as EDTA; buffers, such as acetates,citrates and phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose. Liquid preparations can be enclosed inampules, disposable syringes or multiple dose vials made of glass,plastic or other suitable material. Suitable carriers may includephysiological saline or phosphate buffered saline [PBS], and thesuspensions and solutions may contain thickening and solubilizingagents, such as glucose, polyethylene glycol, and polypropylene glycoland mixtures thereof. Liposomal suspensions, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art.

These compositions that are formulated as solutions or suspensions maybe applied to the skin, or, may be formulated as an aerosol or foam andapplied to the skin as a spray-on. The aerosol compositions typicallycontain [by weight] from 25% to 80%, preferably from 30% to 50%, of asuitable propellant. Examples of such propellants are the chlorinated,fluorinated and chlorofluorinated lower molecular weight hydrocarbons.Nitrous oxide, carbon dioxide, butane, and propane are also used aspropellant gases. These propellants are used as understood in the art ina quantity and under a pressure suitable to expel the contents of thecontainer.

Solutions, may be formulated as 0.01%-10% isotonic solutions, pH about5-8, with appropriate salts, and preferably containing one or more ofthe compounds herein at a concentration of about 0.1%, preferablygreater than 1%, up to 50% or more. Suitable mild solutions are known[see, e.g., U.S. Pat. No. 5,116,868, which describes typicalcompositions of ophthalmic irrigation solutions and solutions fortopical application]. Such solutions, which have a pH adjusted to about7.4, contain, for example, 90-100 mM sodium chloride, 4-6 mM dibasicpotassium phosphate, 4-6 mM dibasic sodium phosphate, 8-12 mM sodiumcitrate, 0.5-1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride,15-25 mM sodium acetate, 10-20 mM D.L.-sodium β-hydroxybutyrate and5-5.5 mM glucose.

The active materials can also be mixed with other active materials, thatdo not impair the desired action, or with materials that supplement thedesired action.

(4) Gels

Gel compositions can be formulated by admixing a suitable thickeningagent to the previously described [(3)] solution or suspensioncompositions. Examples of suitable thickening agents have beenpreviously described with respect to the lotions.

The gelled compositions contain an effective amount of one or more ananti-hyperalgesic amount, typically at a concentration of between about0.1 mg/l-10 mg/l or more of one or more of systems provided herein, from0% to 75%, from 0.5% to 20%, preferably from 1% to 10% of the thickeningagent; the balance being water or other aqueous carrier.

(5) Solids

Compositions of solid forms may be formulated as stick-type compositionsintended for application to the lips or other parts of the body. Suchcompositions contain an effective amount of one or more of the compoundsprovided herein. The amount is typically an amount effective to glowwhen contacted with moist skin, such as lips, typically at aconcentration of between about 0.1 mg/l-10 mg/l or more of one or moreof the systems provided herein. The solids also contain from about 40%to 98%, preferably from about 50% to 90%, of the previously describedemollients. This composition can further contain from 1% to 20%,preferably from 5% to 15%, of a suitable thickening agent, and, ifdesired or needed, emulsifiers and water or buffers. Thickening agentspreviously described with respect to lotions are suitably employed inthe compositions in solid form.

Other ingredients, such as preservatives, including methyl-paraben orethyl-paraben, perfumes, dyes or the like, that are known in the art toprovide desirable stability, fragrance or color, or other desirableproperties, such as shielding from actinic rays from the sun, tocompositions for application to the skin may also be employed in acomposition for such topical application.

2. Glowing toys and other items

Examples of uses of the bioluminescence generating systems in toysinclude illumination of dolls, toy vehicles, hoolahoops, yo-yos,balloons, immersible bubble generating toys, such as a toy submarinethat blows glowing bubbles, and any other toy amenable to having agenerally translucent covering defining a space for containment of thebioluminescence generating system and addition of the final ingredientsnecessary for the illumination reaction. Also contemplated herein aretoys that eject or spew a fluid. For example, toy or game projectilesare contemplated that contain a luciferase and bioluminescence substratein an oxygen-free environment. The projectiles rupture upon impact witha hard surface thereby exposing the contents to moisture in the air thatcontains dissolved oxygen, the bioluminescence activator, and causingreaction.

Dolls and dummies containing one or two of the bioluminescencegenerating system components within a transparent or translucent portionof their bodies are also contemplated herein. Addition of the remainingbioluminescence generating system component(s) results in illuminationof that body part or area. For example, a doll can have a visible,translucent digestive system containing a luciferase and substrate in awater-free environment. Upon "ingestion" of water by the doll, that isaddition of water through its mouth, for example, the digestive systemglows or is illuminated.

Other examples of uses of the bioluminescence generating systems in toysinclude, but are not limited to illuminated hoolahoops, yo-yos, slimyplay materials, such as those based on sodium alginate and glycerine[U.S. Pat. No. 5,310,421], such as those sold by MATTEL® as FLOAM®,GAK®, and SMUD® and moldable play materials, such as those described inU.S. Pat. Nos. 2,541,851, 3,384,498, 3,565,815, 3,634,280, 3,661,790,3,804,654, 3,873,485, 4,076,547, 4,172,054, 4,229,790, 4,624,976 and4,735,660, all of which are incorporated herein in their entirety. Withrespect to the slimy and moldable play materials, the bioluminescencegenerating components can be incorporated into the play material duringmanufacture, as liposomes, or linked to the material.

In one embodiment, the slimy play materials are fabricated from selfcross-linking sodium alginate, a glycerin solution [concentration over90%], water and preservatives.

In an alternative embodiment, the slimy play materials are fabricatedfrom polyvinyl alcohol and tetraborate. In another embodiment, discussedfurther below and in the Examples, the slimy play material is packagedin a compressible dispensing apparatus, for example, as illustrated inFIG. 27. In such an apparatus, all but one of the bioluminescencegenerating reagents may be provided in a compartment situated within thedispensing apparatus. A second compartment within the apparatus maycontain less than all the components required to complete the slimy playmaterial composition, and the main body of the apparatus may contain theremaining bioluminescence generating reagents and/or remaining slimyplay material components.

Alternatively, three compartments within the compressible dispensingapparatus may be provided where, the third compartment contains eitheror both of the remaining bioluminescence generating reagents or theremaining slimy play material components. The main body of the apparatuswould then contain an aqueous composition within which to mix thecontents of the three compartments or the bioluminescence generatingreagents or slimy play material components not contained within thethird compartment.

In still other embodiments, the slime material is provided withoutbioluminescence generating reagents and the bioluminescence generatingreagents are provided as separate compositions, in time release vehiclesor other delivery vehicles, and are mixed into the material prior touse.

Another slimy material provided herein is prepared from 2-4% sodiumtetraborate 2-3 ml and 2-8% polyvinyl alcohol mixed with 10 ml add 100μgs charged aequorin or other suitable luciferase. When used withaequorin, addition of a little water [tap water or othercalcium-containing aqueous medium] results in slime material that lightsup. As mentioned above, one embodiment of an apparatus designed forcontaining and delivering the slimy play material is shown in FIG. 27.The apparatus is a compressible apparatus, for example, like atoothpaste tube, having one, two or three, preferably two, compartmentsinside the compressible apparatus. The compartments are formed, at leastin part, of a readily rupturable material, such as plastic, such thatupon squeezing the compressible apparatus, the contents of thecompartments are released into the main body of the apparatus and arethereby mixed.

One compartment of the compressible apparatus may contain slime materialwith a luciferase and the other compartment contain the remainingbioluminescence generating components or the remaining components inslime. Alternatively, one compartment contains sodium tetraborate andluciferase and the other compartment contains the polyvinyl alcohol. Ina three compartment system, one compartment may contain luciferin andluciferase packaged in the absence of oxygen. The second compartment maycontain the polyvinyl alcohol and the third compartment contain thesodium tetraborate. The main body of the compressible apparatus wouldthen contain the remaining slime material ingredients and the remainingbioluminescence generating reagents, such as calcium ion. If oxygen isthe final bioluminescence generating reagent required, it may be presentin the aqueous slime material composition present in the main body ofthe apparatus, or it may be provided by the atmosphere when the slimematerial is expelled. Other variations in which the components areseparated are also contemplated herein.

Other alternative embodiments of the moldable play materials includethose fabricated from dimethyl silicone treated with a compound of boronpreferably followed by further treatment using heat and/or a catalyst,as described in U.S. Pat. No. 2,541,851; those fabricated frommanogalactan gum, alkali metal borate, boric acid, high molecular weightpolysaccharide, bacteriostat, fungistat, filler, colorant and perfume,as described in U.S. Pat. No. 3,384,498; those fabricated from materialfillers, such as clay and talc, together with hydrocarbon petroleumdistillate oil, waxy paraffinic hydrocarbon oil, a liquid siliconecompound, an astringent, a humectant, glue and water, such as describedin U.S. Pat. No. 3,804,654; those fabricated from synthetic resin and awooden powder together with an oil formulation, where the syntheticresin is a rubber reinforced styrene resin and the oil used is ahydrocarbon oil utilizing an aromatic ring forming carbon, such asdescribed in U.S. Pat. No. 4,624,976; or those fabricated from woodflower combined with a water-based gel using cross-linkable guar gum asa gellant, such as described in U.S. Pat. No. 4,735,660.

The glycerin based slimy play materials, such as those described in U.S.Pat. No. 5,310,421] contains 2.5-4.0 by weight 3.33 weight %, of aself-crosslinking sodium alginate; 1.0-3.5 weight % of a glycerin andwater composition in excess of 90% glycerin; a preservative; 4.0 weight% NaCl; and water, and can include 0.04-0.08 weight % of a colorant. Asmodified herein, it will also include up to all but one component of abioluminescence generating system, such as a luciferase, such as Renillaor Vargula or a firefly luciferase, or a luciferin and luciferase, suchas the Aequorin photoprotein and EDTA. A second mixture of the slimematerial will contain the remaining components.

A preferred slimy material contains 2.5-4.0% by weight, preferably 3.33%by weight, of a self-crosslinking sodium alginate; 1.0-3.5% by weight ofa glycerin and water solution in excess of 90% glycerin; one or morepreservatives; 2.0-7.0%, preferably about 4%, by weight NaCl; and water,and can include 0.04-0.08% by weight of one or more colorants. Thematerial will also include up to all but one component of abioluminescence generating system, such as a luciferase, such as Renillaor Vargula or a firefly luciferase, or a luciferin and luciferase, suchas the Aequorin photoprotein and EDTA.

The slimy play material may be made to glow by contacting it with asecond mixture of the slime material containing the remaining componentsof the bioluminescence generating system or by contacting it with theair or an aqueous composition, where molecular oxygen or calcium ion isrequired to complete the reaction. The second mixture can also contain adifferent colorant, so that upon mixing not only will the material glow,it will change color.

The concentrations of bioluminescence system components, such asluciferase, will be those sufficient to generate a visible glow. Theconcentrations of luciferase can be empirically determined, butgenerally will be between about 0.1 and 1 mg per liter of material. Theamount of luciferin generally will be in excess. The luciferases andluciferin and other components can also be provided as time releasevehicles in the material or provided separately for subsequent addition.

This slime material can be packaged as a kit or article of manufacturecontaining a first slime composition containing all but at least onebioluminescence generating reagent, and a second slime compositioncontaining the remaining components. The kit will include instructionsfor mixing the two compositions to produce a glowing composition. Thekit can also contain additional compositions or vehicles or driedpowders of bioluminescence generating reagents so that they can be addedprior to use so that the material can be reused.

In another embodiment, discussed further below and in the Examples, theslimy play material is packaged in a compressible dispensing apparatus,for example, as illustrated in FIG. 27. In such an apparatus, up to allexcept for one of the bioluminescence generating reagents may beprovided in a compartment situated within the dispensing apparatus. Asecond compartment within the apparatus may contain less than all thecomponents required to complete the slimy play material composition, andthe main body of the apparatus may contain the remaining bioluminescencegenerating reagents and/or remaining slimy play material components.

Alternatively, for example, three compartments within the compressibledispensing apparatus may be provided such that the third compartmentcontains one or all of the remaining bioluminescence generating reagentsor the remaining slimy play material components. The main body of theapparatus would then contain a composition, typically an aqueoussolution within, which to mix the contents of the three compartments orthe bioluminescence generating reagents or slimy play materialcomponents not contained within the third compartment.

In still other embodiments, the slime material is provided withoutbioluminescence generating reagents and the bioluminescence generatingreagents are provided as separate compositions, in time release vehiclesor other delivery vehicles, and are mixed into the material prior touse.

Another slimy material provided herein is prepared from 2-4% sodiumtetraborate 2-3 ml and 2-8% polyvinyl alcohol mixed with 10 ml add 100μgs charged aequorin or other suitable luciferase. When used withaequorin, addition of a little water [tap water or othercalcium-containing composition] results in slime material that lightsup. As mentioned above, one embodiment of an apparatus designed forcontaining and delivering the slimy play material is shown in FIG. 27.The apparatus is a compressible apparatus, for example, like atoothpaste tube, having one, two or three, preferably two, compartmentsinside the compressible apparatus. The compartments are formed, at leastin part, of a readily rupturable material, such as plastic, such thatupon squeezing the compressible apparatus, the contents of thecompartments are released into the main body of the apparatus and arethereby mixed.

One compartment of the compressible apparatus may contain slime materialwith a luciferase and the other compartment contain the remainingbioluminescence generating components or the remaining components inslime. Alternatively, one compartment contains sodium tetraborate andluciferase and the other compartment contains the polyvinyl alcohol. Ina three compartment system, one compartment may contain luciferin andluciferase packaged in the absence of oxygen. The second compartment maycontain the polyvinyl alcohol and the third compartment contain thesodium tetraborate. The main body of the compressible apparatus wouldthen contain the remaining slime material ingredients and the remainingbioluminescence generating reagents, such as calcium ion. If oxygen isthe final bioluminescence generating reagent required, it may be presentin the aqueous slime material composition present in the main body ofthe apparatus, or it may be provided by the atmosphere when the slimematerial is expelled. Other variations in which the components areseparated are also contemplated herein.

Other toys, games, novelty items, clothes, accessories, foods,beverages, fountains, water dispensing apparatus, soaps, creams,cosmetics and sporting equipment amenable to bioluminescence are furtherembodiments of the presently disclosed combination. Thus, any article ofmanufacture or substance capable of modification to allowbioluminescence thereof is contemplated herein.

Articles of manufacture that are amenable to use with thebioluminescence generating systems provided herein are well known [see,e.g., U.S. Pat. Nos.: 5,415,151, 5,018,449, 3,539,794, 5,171,081,4,687,663, 5,038,963, 4,765,510, 4,282,678, 5,366,108, 5,398,827,5,397,014, 5,219,096, 5,305,919, 5,184,755, 5,029,732,4,214,674,4,750,641, 4,676,406], which describe devices useful as toywater guns or vessels for beverages or creams and lotions. To beamenable to use in the embodiments described herein, each may requiresome modification, such as, for example, addition of a mixing chamber.

In light of the disclosure herein, such modification will be apparent.Some of the patents describe other toy devices, training mock weapondevices, dolls, and beverage containers and dentifrice containers [i.e.,toothpaste tubes]. In the simplest modification, powdered or capsularvehicles containing bioluminescence generating systems may be added tothe water-holding chambers of the toy gun or other water spewing toy. Asthe powder dissolves or the vehicle releases its contents, typicallyluciferin and luciferase, contact with the water in the gun will causethe bioluminescence reaction to occur.

As is apparent from the above, toy guns are well known items andmaterials and specifications for manufacture thereof are also well known[see, the above list and see, also, U.S. Pat. Nos. 5,029,732, and5,415,151]. Any single chamber squirt gun may used in combination withbioluminescence generating systems herein by mixing the components inthe gun chamber. Of course the selected system should be one that hassustained illumination. Alternatively, pellets of encapsulatedbioluminescent components, such as the aequorin photoprotein or theRenilla luciferase and luciferin, may be added to water in the gunchamber. In the case of the aequorin photoprotein and Renillaluciferase, added tap water may be sufficient. For the Renilla systemthe pellets may contain the luciferase and luciferin or either. Theremaining component will be added to the gun chamber. If pellets areused, the pellets will slowly release their contents thereby providingfor a continuous glow.

Similar apparatus and designs are also used for any fountain or waterpropelling device. Any such device [see, e.g., U.S. Pat. No. 5,360,142]may be modified to include a bioluminescence system to produce a glowingstream.

In all of these devices, the water, for example, can be tap water or aselected buffer, particularly phosphate buffered saline. The items maypackaged as kits with the packaged luciferin, luciferase, and includingthe water.

a. Single chamber toy guns and other toy weapons that shoot pellets orliquid

Numerous toy guns and other toy weapons that shoot pellets or liquid, inaddition to those exemplified herein, are suitable for use incombination with the bioluminescence generating systems herein. The toyweapons may be loaded with a composition containing microspheres ofluciferin and/or luciferase, or with lyophilized luciferin/luciferin, orother mixtures as described herein. Suitable toy weapons and devicesthat shoot jets or sprays of water are described in the followingsampling of U.S. Pat. No. 5,462,469 [toy gun that shoots bubbles]; U.S.Pat. No. 5,448,984 [toy gun that shoots balls and water and can bemodified to shoot light or temperature sensitive pellets, which shouldbe stored under appropriate conditions or appropriately packaged, thatrelease luciferin/luciferase when exposed to light]; U.S. Pat. Nos.5,439,139; 5,427,320; 5,419,458; 5,381,928; 5,377,656; 5,373,975;5,373,833 and 5,373,832 [which describe toy guns that rely upon apressurizable bladder for release of air pressure to shoot a projectile,which can be modified to shoot projectiles of encapsulatedluciferin/luciferase]; U.S. Pat. No. 5,370,278 [which describes liquidfrom a port mounted to a headband]; U.S. Pat. Nos. 5,366,108; 5,360,142[which describes a supply and delivery assembly for use in combinationwith a pump type water gun or other water propelling device]; U.S. Pat.Nos. 5,346,41 8; 5,343,850 [which describes a projectile launcher foruse in combination with the pellets provided herein]; U.S. Pat. Nos.5,343,849; 5,339,987 [which describes water guns that have at least onepressurizable air/ water storage tank, a pressurizing mechanism, achannel of release for shooting water and a release mechanism]; U.S.Pat. Nos. 5,326,303; 5,322,191; 5,305,919; 5,303,847 [which describes adevice worn on a user's hand with sheaths for the tips of the fingersthat includes a housing for a water reservoir, a water pump andelectrical motor and a battery pack to be secured to the user's body];U.S. Pat. Nos. 5,292,032; 5,284,274 [which describes an action to systemincluding a capsule for containing water, which will herein containcomponents of a bioluminescence generating system, having an orifice anda plunger and a spring loaded mechanism for driving the water from theorifice. The action toy may be configured as a shotgun accepting aplurality of prefilled shell capsules into its breechblock for firingthrough its barrel, as a missile launcher in which the capsules aremounted to the front of the launcher and the water is ejected directlyfrom the capsule against the target, or as a crossbow with the bowloading the spring-loaded mechanism and a water stream obtained onrelease of the bow]; U.S. Pat. No. 5,284,272 [which describes a bottleand cap combination for spewing liquid]; U.S. Pat. Nos. 5,256,099;5,244,153; 5,241,944; 5,238,149; 5,234,129; 5,224,625; 5,213,335;4,854,480; 5,213,089; 5,184,755; 5,174,477; 5,150,819; 5,141,467;5,141,462; 5,088,950; 5,071,387 [which describes a figurine-shaped watersquirting toy]; U.S. Pat. No. 5,064,095 [which describes a water cannonapparatus]; U.S. Pat. Nos. 5,029,732; 5,004,444; 4,892,228; 4,867,208[which describes an apparatus for storing and dispensing fluid underpressure]; U.S. Pat. Nos. 4,808,143; 4,784,293, 4,768,681; 4,733,799;4,615,488 and many others. U.S. Patent No. 5,415,151 describes a toy gunthat launches projectiles that can be adapted for shooting the pellets,such as light sensitive pellets that are degraded upon exposure tolight, provided herein.

b. Bubble-making toys

Soap bubbles are blown from water solutions or other aqueous compositioncontaining soap or another surfactant. A great variety of bubbleformulations are available, including those that feature special effectsin bubble making. There are solutions for making large bubbles, "longlasting" bubbles, split bubbles, self-healing bubbles, multiple bubbles,vanishing bubbles, flaking bubbles, bursting bubbles, high and/orfar-flying bubbles, sinking bubbles etc. In general, many anionic,non-ionic or amphoteric aqueous solutions with low surface tension aresuitable for bubble or foam-making when air or other gases are blowninto such compositions.

Such compositions, preferably those that have near neutral pH, can becombined with the components of the bioluminescence generating systemsprovided herein. In particular, a mixture of luciferase and luciferin,such as the Renilla system or firefly system or Cypridina system,preferably in the form of pellets or microspheres, such as liposomes orother time release capsule, can be added to the bubble mixture. Whenused, the air added to the mixture will cause a glow, or a glow will beproduced as the contents of the pellets are released into thecomposition. Alternatively, one or more component of the bioluminescencegenerating system may be added to the bubble making composition, suchas, for example, a luciferase and any necessary activators, and theremaining component(s), e.g., a luciferin, may be directly applied tobubbles using a fine spray from an atomizer or other suitable spray ormisting means.

In addition, a fluorescent protein, such as GFP, BFP or aphycobiliprotein, may be added to the bubble-making composition and thenilluminated using an external light source. For example, bubblescontaining a fluorescent protein may be produced in a room illuminatedwith light of an appropriate wavelength to cause the fluorescent proteinto fluoresce.

Alternatively, the fluorescent protein may be added to the bubble-makingcomposition containing all the components of the bioluminescencegenerating system to effect a change of the color of the bubbles. Forexample, the fluorescent proteins may be added to the bubble-makingcomposition directly or may be added in time-released orslowly-dissolving microspheres or liposomes, such that release of afluorescent protein in the bubble composition, such as, for example, GFPor a phycobiliprotein, introduces a change in the color of the bubbles.It is particularly advantageous to have the fluorescent protein releasedinto the composition after the container has been opened and used. Asingle bottle of bubble-making solution will be amenable to theproduction of more than one color of bubbles. For example,microparticles or liposomes suspectible to breakdown by exposure to airor by agitation by the wand or stick used for blowing bubbles are ofparticular interest.

Kits containing such soap compositions, with preferably a moderate pH[between 5 and 8] and bioluminescence generating reagents, includingluciferase and luciferin and the fluorescent protein are providedherein. These kits, for example, can be used with a bubble-blowing orproducing toy. These kits can also include a reloading or chargingcartridge, such as the cartridges provided herein.

Toys that produce bubbles include bubbles with wand for blowing,bicycles, flying toys, dolls, swords, toy musical instruments, bubblebeards, and numerous other toys are well known [see, e.g., U.S. Pat.No.: RE 32,973, which describes a toy bubble-blowing lawn mower; U.S.Pat. No. 4,511,497, which describes a non-toxic non-irritating bubblecomposition for toys, U.S. Pat. Nos. 2,579,714; 5,480,334; 5,041,042;5,478,267; 5,462,469; 5,419,728; 5,393,256; 5,366,402; 5,348,507;5,322,464; 5,304,085; 5,269,715; 5,224,893; 5,183,428; 5,181,875;5,156,564; 5,135,422; 5,080,623; 5,078,636; 4,957,464; 4,955,840;4,943,255; 4,923,426, 4,867,724; 4,861,303; 4,840,597; 4,808,138;4,804,346; 4,764,141; 4,700,965; 4,556,392 4,334,383; 4,292,754;4,246,717; and many others].

c. Board/Card Games

Board games, card games and similar entertainment items may be used incombination with the bioluminescence generating systems describedherein. The boards or cards may be constructed of paper or fabric, asdescribed herein, or may be constructed of plastic or other polymeramenable to covalent or non-covalent attachment of bioluminescencegenerating compontents.

A particular portion of the game board or a card piece is covered orimpregnated one or more up to all but one of the bioluminescencecomponents. A developing wand or sponge or similar apparatus isimpregnated or coated or dispenses the remaining bioluminescencecomponent(s) [developing reagents]. Contacting, such as by wiping, thecard piece or game board with the developing wand or sponge or contentsof the dispensing apparatus will produce a glow.

The developing reagents can be applied to the developing wand or spongein various forms. For example, the developing reagents may be insolution or suspension and the sponge or wand soaked in the solutionthen sealed in an air-tight packaging to be opened immediately beforeuse. Alternatively, the developing reagents may be lyophilized ordessicated and applied in powder form to the wand or sponge. immediatelybefore use, water is added to the wand or sponge and then wiped on thegame board or card piece.

Alternatively, the board and pieces may include adsorbed or absordedlyophillized bioluminescence-generating reagents. Contacting these itemswith water, containing the appropriate salts and buffers, such ascalcium, if for example, the aqueorin system is used, or ATP if thefirefly system is used.

The bioluminescence components applied to the game board or card piececan be applied in a particular pattern, for example to spell a word orillustrate an instruction. Preferably, the bioluminescence system chosenwill be capable of repeated use. For example, the Renilla system, isamong the preferred systems. The luciferase can be linked to the pieces,and the luciferin can be applied to the board or card and a newdeveloping wand or sponge used each time the game is played.

Alternative embodiments will be appreciated, for example, the game canbe an educational one in which the player uses the developing wand orsponge to reveal the correct answer to a question. Similarly, the gameboard may be a puzzle where a "hidden" illustration or message isrevealed by wiping the completed puzzle with the developing wand orsponge.

d. Toy "Eggs" or other encapsulated items

Egg-shaped (or any other desired shape) toys containing a liquid orpaste that glows upon exposure to ambient air are a further example of acombination contemplated herein. The ingredients of the egg compositioninclude a luciferin and luciferase, such as the Cypridina or Vargulaluciferin and luciferase, which requires oxygen for activation. Theliquid or paste is introduced into the "eggs" the eggs are sealed undernitrogen or other suitable gas, other than oxygen or air. Upon exposureto air, by opening or cracking the egg, the egg composition glows. Thisprinciple can be adapted to other uses, such as sphere shapedmacrocapsules that may be shot from a toy gun and burst upon impact, ina manner similar to paint ball guns currently used to shoot paint ballsat targets for marking. In practice, water is de-oxygenated, for exampleby bubbling argon or nitrogen gas through it. The de-oxygenated water isthen used to mix the bioluminescence generating components, other thanmolecular oxygen. The mixing should take place under strictly conditionsin which air or oxygen is excluded, such as in a hood under nitrogen, inorder to prevent exhaustion of the bioluminescence-generatingcomponents.

In one embodiment, to produce a realistic egg-like mixture,approximately 1 to 2 mg of a luciferin/luciferase composition per 30 mlof egg volume is combined with a suitable thickener, such ashydroxymethyl cellulose, to provide the consistency of a real egg. The"shell" of the egg is formed of a suitable material which excludesoxygen (air) and is readily opened by the consumer before use. Forexample, the egg mixture can be packed into paper mache and covered withwax to provide an airtight seal. Similarly, the "shell" may be formedfrom a polymer, such as a plastic, that is airtight but readily brokenwhen desired.

e. Footbags, Bean Bags and Balls

Glowing footbags, bean bags and balls are also provided herein.Footbags, such as the HACKY SACK, which is a registered Trademark ofWham-O Corporation, described in U.S. Pat. No. 4,151,994, are generallyconstructed of an outer leather casing having a diameter of about threeinches, which is filled with small granules, such as beans or othergranular material [see, also U.S. Pat. Nos. 5,429,351, 4,963,117,4,717,158, and 4,002,839]. The sack is used to play a game in whichplayers kick the sack between one another, trying to keep the sack inmotion and off the ground, without using their hands.

Contemplated herein are footbags and balls that glow as they are kickedabout by the players. The bags are fabricated from an inflatabletranslucent material, such as a plastic, Similar to the egg mixturedescribed above, the granules in the footbag are made in an oxygen freeenvironment and packaged such that air/oxygen is excluded until the sackis in use. For example, the granules are made of a gelatinized mixtureof bioluminescence generating system components excluding molecularoxygen and are packaged in an oxygen free package, such as dry nitrogenpackaging, commonly used in marine electronics, or in rupturableliposomal pellets.

The granules can be covered in a flexible plastic of varying thicknessesto allow for the timed ingress of oxygen across the plastic membrane. Asthe footbag is repeatedly kicked by the players, the mechanical stresson the granules allows more oxygen to react with the bioluminescencegenerating components contained therein, creating more light.

An alternative embodiment contemplated herein involves partitioning thegranules within the footbag using, for example, a semi-permeablemembrane material that permits slow permeation of the compositionscontained in the two compartments thereby formed. One compartment isthen filled with all but one or more bioluminescence components and theother compartment is filled with the remaining components. As thefootbag is kicked about, the mechanical stresses on the separatingmembraned force the contents of the two compartments to mix, therebyproviding flashes of light or periods of illumination followed bynon-illumination. For example, in one compartment, a calcium containingcomposition can be added to the beads, and in the other compartment, acoelenterazine-charged aequorin is added. When the footbag is kicked,flashes of light are produced.

The covering of the footbag must be translucent, transparent or somecombination thereof to allow the light generated to be visible. Thus,the "sack" can be formed from clear nylon webbing, translucent ortransparent pliable plastic, translucent or transparent cloth or similarmaterial.

f. Figurines

Glowing figurines are also provided herein. Figurines may be of any sizeor shape and preferably contain at least one chamber that holds liquid.The figurine may be cast, molded or manufactured from any suitablematerial. Preferably a portion of or the entire figurine is translucentto the wavelength of light produced in the bioluminescence generatingreacton. The figurine may be in any design or theme, such ascharacterizations of entertainment and sport celebrities, memorabilia,slogans and logos, trademarks or other promotional items, animals,Christmas ornaments or other inanimate objects. For example, smallfigurines may be placed in areas of dim lighting, e.g., on tables inrestaurants, that contain one or more component of the bioluminescencegenerating system, such as a luciferase. The remaining components of thebioluminescent reaction, i.e., a luciferin and any necessary activators,are added at the desired time and the figurine glows.

In another embodiment, one or more component(s) of the bioluminescencegenerating system may incorporated into or linked to the material fromwhich the figurine is fabricated. The remaining components of thebioluminecsent reaction may be sprayed or applied to the surface of thefigurine to initiate the bioluminescent reaction.

3. Glowing textiles and paper products

The bioluminescence generating systems described herein are alsocontemplated for use with textiles and paper. One or two of thebioluminescence generating system reagents are applied to the textile orpaper and the remaining components are added when illumination isdesired. For example, the luciferase in association with thebioluminescence substrate may be applied to the textile or paper,through covalent or non-covalent interaction. When water, or otherappropriate activator, is applied to the material, illumination ensues.Examples of uses for the textile include the fabric portion of anumbrella, clothing, towels, the fabric portion of artificial plants orflowers, toys having a fabric component or any item susceptible tomanufacture from textile material.

With respect to paper, the luciferase may be applied to the paper inassociation with the bioluminescence substrate. The paper glows uponaddition of the bioluminescence activator to the paper. Thus, if thebioluminescence activator is water, addition of water to the paper, forexample as an aerosol, produces a glow on the paper. The paper may alsobe illuminated by "writing" upon it with one or two of thebioluminescence generating system components then "writing" or sprayingover those components with the remaining component(s). As with the othersystems disclosed herein, the critical aspect to operation ismaintaining at least one of the bioluminescence generating systemcomponents separate from the other components until illumination isdesired. The paper may be in almost any form or of almost any type, suchas writing paper, wrapping paper, boxes, poster paper, books, paperjewelry, paper towels, napkins or other paper products.

4. Foods and beverages, including ice cubes

Examples of beverages and foodstuffs amenable to combination withbioluminescence systems include, but are not limited to, alcoholicbeverages, as well as sodas and juices, and such foods as applesauce andmashed potatoes. Further, bioluminescence generating systems can bechosen and adapted for use in such foodstuffs as cakes and ice creams oralmost any other edible item. Considerations in combiningbioluminescence systems with food and/or beverages are primarily thestability of the system throughout processing of the food or beverage,unless the system is added subsequent to any such processing; theability to contact the system with its finally required ingredients toproduce bioluminescence; and taste of the components of the system withthe foodstuffs to which they are added.

Bioluminescent food products are also contemplated herein. Suchproducts, amenable to combination with the bioluminescence generatingsystems described herein, include those that may be stored between about0° C. and 35° C. Generally, once the luciferase or bioluminescencesubstrate is added to the food product, it cannot be heated above about100° C. Thus, food products requiring cooking prior to consumption alsocan be cooked prior to addition of either the luciferase orbioluminescence substrate.

Examples of food products amenable for use in combination with thebioluminescence generating systems described herein include, but are notlimited to, icings and other toppings or sauces, cookies, biscuits, andsimilar prepared foods. Bioluminescent icings, for example, may beprepared by including the luciferase and bioluminescence substrate in adehydrated icing mixture. Addition of water, just prior to use causesthe mixture to glow. Alternatively, the bioluminescence activator andeither the luciferase or bioluminescence substrate may be included inthe prepared icing mixture and the absent bioluminescence generatingsystem component stirred into the icing just prior to use.

Alternatively, food products may be produced to include a fluorescentprotein, such as a phycobiliprotein or a green or blue fluorescentprotein, and then illuminated using an external light source. Forexample, icing containing fluorescent protein may be served in a roomilluminated with light of an appropriate wavelength to cause thefluorescent protein to fluoresce. Similarly, a fluorescent protein maybe included in an ice cream mixture, in an ice cream topping sauce, in asalad dressing, in cakes, puddings or similar food product and the foodthen subjected to an external light source of appropriate wavelength toinitiate the fluorescence.

a. Beverages

Beverage products are likewise contemplated for use herein incombination with the bioluminescence generating systems describedherein. As with other embodiments, at least one of the bioluminescencegenerating system components is excluded from the beverage untilbioluminescence is desired. For example, a container/bladder apparatus,as described generally above and in detail below, maintains theluciferase and bioluminescence substrate separate from the beverage.Upon opening of the container, the luciferase and substrate are added tothe beverage causing it to glow.

Alternatively, the beverage may be produced and packaged alreadycontaining one or two of the bioluminescence generating systemcomponents, such that addition of the remaining components causes aglow. An example of such a beverage is bioluminescent beer, wine,champagne or a soft drink. In this embodiment, the yeast used to producethe alcohol component of the beer or other beverage, are geneticallytransformed to contain, for example, a gene encoding a luciferase andthe complementary genes necessary to direct the yeast to manufacture andsecrete the luciferase. Assuming O₂ or air is the bioluminescenceactivator, then when a glow is desired, the bioluminescence substrate isadded to the beer.

Another example of a bioluminescent beverage contemplated herein is asoft drink containing two of the three bioluminescence generating systemcomponents. When bioluminescence is desired, a third bioluminescencegenerating system component is added. If the bioluminescence generatingsystem is, for example, the Aequorin system or the Renilla system, thenthe Aequorin luciferase with bound luciferin or the Renilla luciferaseand the luciferin may be included in the soft drink and thebioluminescence activator, Ca²⁺ [for the aequorin system] or dissolvedO₂, added to the beverage to cause a glow. Suitable vessels for suchbeverages are provided herein [see, EXAMPLES] and also are known tothose of skill in the art [see, e.g., U.S. Pat. No. 5,398,827].

Similarly, a soft drink beverage can be produced containing all thebioluminescence generating system components except, for example,dissolved oxygen where the bioluminescence generating selected requiresoxygen to complete the bioluminescent reaction. In lieu of carbondioxide, the beverage may have another gas or gasses dissolved therein,for example nitrogen, helium, nitrous oxides or helium oxygen (heliox).The soft drink is packaged under oxygen free conditions and, uponopening of the soft drink container and exposure of its contents to theair, the oxygen in the air activates the bioluminescent reaction causingthe soft drink to glow.

In each of the above embodiments, it is also contemplated thatslowly-dissolving or time releasing microparticles, such as, but notlimited to liposome or isolated endosomes, may be included in thebeverage that contains additional bioluminescent components.Microparticles may contain, for example, one or more luciferases, aphycobiliprotein, a green or blue fluorescent protein, a luciferin orany mixture or combination thereof. Upon dissolution of themicroparticle or release of the contents by other means, the contents ofthe microparticle are released into the beverage or other liquid,resulting, for example, in a change in the color of the emitted lightthe beverage, an change in the color of the bioluminescent light and/oran increase in the intensity of the emitted light of the entire beverageor just a portion thereof. By selecting the appropriatemicroparticle(s), the release of one or more component of the reactionmay be effected sequentially or concurrently. Thus, drinks in whichseveral glowing colors are produced are contemplated herein. Multiplecolor changes are effected by the appropriate selection ofbioluminescence generating agents and/or fluorescent proteins.

For example, an appropriate time-released or slowly-dissolvingmicroparticle containing a GFP or a phycobiliprotein may be added to abeverage containing the Renilla or aequorin bioluminescence generatingsystem. Upon dissolution or release of the fluorescent protein into themedium, the initial blue color of the glowing beverage is converted toanother color, e.g., converted to a green color by the GFP. Theinclusion of an additional microparticle containing a phycobiliproteinwith an absortion maxima in the green spectra, in which themicroparticle has been selectively designed to dissolve or release intothe beverage after release of the GFP, would result in the beverage onceagain changing color to, for example, red. The color of the beverage maybe changed sequentially and repeated as many times as desired. Thenumber of possible color changes will depend on the type of beverage,the desired colors and the duration of each color. Any beverage iscontemplated for the color changes as described herein, such as softdrinks, alcoholic beverages, juices and the like.

Alternatively, the color change may be designed to be effected in only aportion of the beverage. For example, microparticles that contain afluorescent protein in combination with a composition that has a higheror lower specific density than the beverage [e.g., a saturated sucrosesolution or any sutiable non-toxic, highly viscous solution having ahigher specific density]. Dissolution or release of the contents of themicroparticle results in the formation of a biphasic solution in which,for example, the top portion of the beverage glows blue whereas thebottom portion of the beverage containing the released fluorescentprotein [e.g., GFP or a phycobiliprotein] glows green, red or anothercolor. The concentration of the fluorescent proteins and the selectionof a higher or lower density liquid and percentages to be used hereinmay be determined empirically by one of skill in the art.

The color of each layer may be changed sequentially or the color changemay be effectively repeated in any order depending on the microparticleor macroparticle employed [e.g., inclusion by direct addition, timereleasing particles or thermal or pH sensitive microparticles].

b. Ice

Ice containing bioluminescent components, such as lyophilized componentsor encapsulated components is contemplated herein. Upon addition to aliquid containing any remaining components or exposure to air, thecontents of the ice will be released as they melt to produce a glow. Theice may be in any shape or form. Examples of ice formations, include butare not limited to, geometric shapes, such as spheres and cubes; iceformations made from precast molds, such as figurines, icicles,popsicles; shaved ice, such as snow cones or imitation snow forrecreational activity like skiing, sledding or snow-mobiling; icesculptures, where the ice glows and/or in combination an inanimateobject frozen within the ice that glows. In addition, ice used as asurface for recreational ice skating or hockey is also contemplatedherein.

The ice may contain one or more of the bioluminescence generatingcomponents. For example, the ingredients of ice may include a luciferinand/or luciferase, such as the Cypridina or Vargula luciferin andluciferase, which requires oxygen for activation. Luciferases isolatedfrom different species that result in the production of light other thangreen or blue, e.g., Aristostomias or Pachystomias which emit red light,or additional components which alter the wavelength of the emittedlight, e.g., a green fluorescent protein or a phycobiliprotein, used inconjunction with the luciferase are also contemplated herein.

In practice, water is de-oxygenated, for example, by bubbling argon ornitrogen gas through it. The de-oxygenated water is used to mix all ofthe bioluminescence generating components besides molecular oxygen. Themixing should take place under strict conditions in which air or oxygenis excluded, such as in a fume hood under nitrogen, in order to preventexhaustion of the bioluminescence-generating components.

The water is placed in a tray, a vessel, a precast form of a particularshape or design, stored or maintained under an inert atmosphere and snapfrozen using liquid nitrogen. The resulting ice is packaged in a sealedcontainer under an inert atmosphere lacking molecular oxygen (e.g.,argon or nitrogen). Upon exposure to air or a liquid containingdissolved oxygen, the ice glows.

Alternatively, one or more component of the bioluminescence generatingsystem may be applied to the surface of the ice to initiate orre-generate the bioluminescent reaction. This method is particularlysuitable for production of a glowing ice surface, such as an ice skatingrink. The components of the reaction may be added to the water containedwithin the Zamboni ice cleaning machine. The water from the machine isoverlayed over the existing ice, which contains (or is first coated onthe surface) at least one component of the bioluminescence generatingsystem, as a thin coating of a composition that contains the other oneor more component(s) of the bioluminescence generating system. As thetwo layers meet, the bioluminescence generating system is produced orrestored and the ice glows.

Furthermore, microparticles containing additional bioluminescencegenerating components may be added to water prior to snap freezing. Forexample, microparticles containing or coupled to a phycobiliprotein or agreen/and or blue fluorescent protein (GFP) can be produced. Theadditional components may also be added to the surface of the ice afterfreezing. As with the beverages, described above, as the microparticlesdissolve in the ice or as the ice melts, the fluorescent protein orother components are released. The presence of the fluorescent proteinconverts the wavelength of the light emitted from the surface orinterior of the ice, which can include the components of abioluminescence generating system, thereby changing the color of the iceor liquid, for example, from blue to green or red. The addition of GFPalso increases the intensity of the green light emitted about 2-5-fold.Thus, a beverage containing such ice would not only change color as timeproceeds but also glow more brilliantly. The light insenity of theliquid could also be enhanced by the addition of microparticlescontaining an appropriate luciferin or activator that upon dissolvingwould provide additional substrate to promote the bioluminescentreaction.

The components may also be combined with dry ice, which as it sublimes,will release the components that contact with moisture condensing in theair. This will produce a glowing fog for use, for example, in theatricalproductions.

c. Other foods

Other foods contemplated herein include a transgenic corn that expressesluciferase. The corn is served with butter containing luciferin,particularly coelentrazine, and as result glows.

Alternatively, the popcorn is engineered to express a fluorescentprotein. When exposed to the appropriate light, the corn will glow.

5. Jewelry, Clothing and Other Items of Manufacture

The bioluminescence generating systems and fluorescent proteins can beused in combination with articles of manufacture that include jewelry,clothing, figurines and other such items. In particular, these items maybe manufactured from matrix materials or from mixtures of the matrixmaterial and other materials. Alternatively, the matrix material may becoated on or impregnated in such articles. Bioluminescence generatingreagents, particularly, luciferases can be linked to the matrixmaterial. When a glow is desired the article can be contacted withcomposition containing the remaining components.

In addition, articles, such as clothing, particularly, T-shirts andsports gear, and paper items may be sprayed with two compositions, thefirst containing less than all of the necessary reagents and the secondcontaining the remaining reagents.

In other embodiments, the article may be made of two vessels separatedby a removable separating means, so that when desired componentscontained therein communicate and react resulting in bioluminescence.

6. Fountains

Numerous fountains and other water spraying apparatus and devices foruse in such apparatus, in addition to those exemplified herein, aresuitable for use in combination with the bioluminescence generatingsystems herein [see, e.g., U.S. Pat. Nos.: 5,480,094; 5,472,140;5,439,170; 5,402,836; 5,388,285; 5,381,956; 5,337,956; 5,288,018;5,167,368; 4,852,801; 3,894,689; 3,889,880; 3,838,816; 3,820,715;3,773,258; 3,749,311]. For use herein, the fountains will be modified oradapted [see, e.g., EXAMPLES] so that jets of liquid containingbioluminescent will spew.

Fountains can be recharged, for example, by adding additional substrateand other activators. Spent substrate should be removed, such as bypassing the water through an affinity matrix specific for the oxidizedsubstrate.

7. Non-Tobacco Cigarettes

Also contemplated herein is a novelty item that is shaped like acigarette and that includes a bioluminescence generating system, whichproduces glowing "smoke" upon exhalation by the user. The usercontemplated herein is an adult former smoker who derived pleasure fromblowing smoke rings. The toy cigarette can be made, for example, byplacing, under oxygen free conditions, a lyophilized, micropulverizedmixture of the bioluiminescence generating system components intoliposomes, as described above, or other packaging material, such asporous plastic microspheres, made from TYGON or other biocompatiblenon-toxic material. The liposomes (or other packaging) are selected tobe of a suitable size to facilitate or permit passage into thebronchioles of the user. The liposomes are preferably on the order of5-10 μM in diameter and are situated in a tubular delivery vehicle [the"cigarette"].

An example of an appropriate delivery vehicle is a thin glass vialsurrounded by plastic, similar to vials known to those of skill in theart that are used for storing amyl nitrate, betadine and benzoinsolutions. The delivery vehicle is preferably shaped and sized like astandard cigarette. The plastic covering is preferably cylindrical witheach end open to allow for the passage of air upon inspiration. Theplastic covering is surrounded by a filter material that allows passageof the liposomes from the device, but prevents the accidental inhalationof particulates, such as glass, if the vial is broken. Additionalfilters, having pore sizes of about 10 μM, are placed at either end ofthe "cigarette" as a further barrier to inhalation of any materiallarger than the liposomes. Solid plastic or similar material caps may besituated over each end of the "cigarette" to prevent the liposomescontained therein from falling out. These caps would be removed justprior to use of the "cigarette", to permit the free flow of air throughthe device. The liposomes are preferably held within the deliveryvehicle by friction.

In operation, the user will inhale the liposomes or similarencapsulating vehicles, which release their contents upon contacting thelungs. The humid environment of the bronchial tree then provides thewater and oxygen necessary to complete the bioluminescence reaction.Upon exhalation, the air leaving the users lungs is illuminated,providing glowing "smoke". If the packaging apparatus chosen for thebioluminescence generating components is a porous plastic microsphere,such as TYGON, then the bronchiolar-ciliary transport mechanism of thebody will transport the spent microspheres out of the bronchia and intothe digestive system. Because plastic is biologically non-reactive, themicrospheres will be passed from the body through normal excretorypathways without eliciting an immune or toxic reaction.

8. Fish and Fish Food and Fish Bait

Also contemplated herein are genetically engineered fish that expressluciferin or, preferably luciferase, and food therefor. Such fish may beproduced may any method known to those of skill in the art forpreparation of transgenic fish. For example, to produce the fish, fisheggs are transfected with a gene encoding a particular luciferase andany other genes or regulatory sequences necessary to direct the fish tomanufacture and express the luciferase, using methods known to those ofskill in the art. Methods for generating transgenic fish are known [see,e.g., U.S. Pat. Nos. 5,512,421, 5,510,099, 5,489,742, 5,476,779,5,416,017 and 5,166,065; see, also, Ozato et al. (1986) Cell Differ.Devel. 19:237-244, Inoue et al. (1990)Cell Differ. Devel. 29:123-128,Rokkones et al. (1989) J. Comp. Phviol.B 158::751-758, and Guyomard etal. (1989) Biochimie 71:857-863, which describe preparation oftransgenic medaka, medaka, salmon and trout, respectively]. Transgenicfish of numerous species have been prepared, providing the skilledartisan with a variety of procedures for developing transgenic fish.Thus, using a transfection methods known to those of skill in the artand methods for introduction and expression of luciferase, transgenicfish that express a luciferase are prepared. Desirably, the fish expressthe luciferase on cell surfaces, such as by incorporating the luciferaseinto DNA encoding a membrane-spanning protein, or express the luciferaseso that it is secreted into the digestive systems or mouths of the fish.

The resulting fish are fed food containing an appropriate luciferin orluciferins [or luciferase] and any additional bioluminescence generatingreagents required. Typically, the luciferin will be present in the fishfood at concentrations ranging from about 1 part per million (ppm) toabout 1 part per 10, weight/weight. As the luciferin, bioluminescentactivators and other system components come in contact with theluciferase expressed by the transgenic fish, the fish or selected organsor tissues will glow. For example, if the luciferase is expressed on thetissues lining the transgenic fish's mouth, then its mouth will light upas it eats the fish food. Similarly, if the fish transfected with theluciferase gene is transluscent, then the digestive organs, particularlythe stomach, will glow as the bioluminescence generating components comeinto contact and complete the bioluminescent reaction. The selectedluciferase/luciferin systems should be one that is resistant toconditions, such as the acidic pH of the digestive system, in the fish.

Thus, for purposes herein, fish food that includes luciferin, preferablyin lyophillized form, particularly, Renilla coelenterazine and Vargulaluciferin, is provided. The transgenic fish that express luciferase orluciferin are also provided.

Also contemplated herein, are glowing fish lures for use as fish bait.

9. Plant food

Plant food, containing a luciferase or luciferin, for use withtransgenic plants that express a luciferin or luciferase. For example,transgenic plants that express a luciferase are known [see, e.g., U.S.Pat. Nos. 5,464,758, 5,436,392, 5,432,081, 5,412,085, 5,362,865,5,268,463, and 5,015,580]. When treated with [i.e., fed] plant foodcontaining a luciferase and other needed components of thebioluminescence generating system, these plants glow.

Plant food containing one or more components of the bioluminescencegenerating system, preferably a luciferin, is provided herein foradministration to transgenic plants that express a luciferase. The plantfood containing a luciferin and any necessary activators may be in theform of any composition that is typically applied to a plant to promoteor maintain growth [e.g., see U.S. Pat. Nos. 4,016,880, 4,711,659,4,804,403, 5,547,486, 5,553,853, RE 35,320, and RE 31,801]. Theluciferin and any activators may be added directly to the plant foodmixture or housed in a separate compartment and added to the plant foodimmediately prior to use. The plant food may be applied to the soil,sprayed on the foliage of the plant or any combination thereof.

F. Cartridges for loading or reloading the novelty items

In order to effectively charge, recharge or refill the bioluminescencegenerating systems that are part of the novelty items, a variety ofcartridges are contemplated herein. It is to be appreciated that anycharging device discussed herein is capable of either initially charginga novelty item, such as a squirt gun, or recharging such a novelty itemonce one or more component(s) of the bioluminescence generating systemis depleted. Exemplary embodiments are set forth in FIGS. 28-34 anddescribed in EXAMPLE 14 below.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1

Dual Chamber Fluid Dispensing Apparatus--Toy Water Gun

An exemplary embodiment of the dual chamber fluid dispensing apparatusis a toy water gun as illustrated in FIGS. 1 through 3. The followingdescription of that preferred embodiment is made with reference to thosefigures. The toy water gun includes two housings [or chambers] 10, 12that conveniently may be constructed of injection-molded plastic orother suitable material. The two housings 10, 12 are affixed, such asglued, heat sealed or by other such means, along a median seam 46 toform the body of the water gun. See especially FIGS. 2 and 3.

In operation, one housing 10 contains a mixture having less than all thecomponents necessary for generating bioluminescence and the otherhousing 12 contains a mixture having the remaining components or theremaining components, save the bioluminescent activator. Depression ofthe trigger 14 pushes the pistons 26, 36 into their respective cylinders38, 48 compressing the trigger springs 28, 43 and pushing the contentsof the cylinder through the second check-valve 34, into the mixingchamber 20 and out the nozzle orifice 22. As the trigger 14 is released,the trigger springs 28, 43 return to their relaxed state pushing thepistons 26, 36 out of the cylinders 38, 48 creating a vacuum thereinwhich pulls the contents of the housings 10, 12 past the firstcheck-valves 33, 32, respectively and into the cylinders 38, 48respectively. Pumping the trigger, that is repeatedly depressing andreleasing it, moves the mixtures contained in the housings through thegun and out the nozzle orifice 22.

As the mixtures leave the cylinders 38, 48, they enter the mixingchamber 20, via the conduit means 44 and second check-valve 34.Luminescence begins either upon mixing of the components or as the mixedcomposition contacts the air upon expulsion from the toy gun. Themixtures may be powdered, such as those produced by lyophilization, orthey may be liquid. If powdered, water can be added prior to use.

The housings 10, 12 may be filled and refilled through the filling caps17, 19, respectively, located at the top of each housing. A trigger 14is attached to a trigger guide 13 which serves to guide the trigger 14towards two piston assemblies 25. Depression of the trigger 14 activatesthe two piston assemblies 25. This causes a portion of the compositionlocated in each housing 10, 12 to move through the water gun into amixing chamber 20 and out a nozzle orifice 22. The preferred embodimentillustrated has a trigger guard 15 which helps prevent accidentaldischarge of the gun and makes the gun appear more realistic. Thesighting aids 21, 23 aid in aiming the toy gun and also serve to makethe gun appear realistic.

Only one of the two piston assemblies 25 is completely illustrated, andit is visible in FIG. 1. The other piston assembly is adjacent to and,in this preferred embodiment, identical to the one illustrated. Theseassemblies operate by substantially identical means and are activated bydepression of the single trigger 14. The piston assembly 25 includes apiston 26 which passes through a sealing o-ring 30, is connected to atrigger spring 28 and moves within a cylinder 38. The piston assemblyalso includes a spring retainer 40 that secures one end of the triggerspring 28 to the end wall of the cylinder. The cylinder 38 is incommunication with one end of a pick-up tube 18 and lies aboutperpendicular to the pick-up tube 18. The cylinder 38 also communicateswith the mixing chamber 20 via conduit means 44.

In the sectional views of the water gun, illustrated in FIGS. 2 and 3,portions of the second, adjacent piston assembly are visible. Namely,the second trigger spring retainer 42 and trigger spring 43 are visiblein FIG. 2, and the second piston 36 is visible in FIG. 3.

Referring to the piston assembly 25 illustrated in FIG. 1, the piston 26passes into the water gun through the sealing o-ring 30 and into thecylinder 38. The trigger spring 28 is attached by one end to the pistonand by its other end to the spring retainer 40 located at the oppositeend of the cylinder from the piston. As the trigger 14 is depressed, thepiston 26 moves into the cylinder 38 and through the sealing o-ring 30.This compresses the trigger spring 28 within the cylinder 38. As thetrigger 14 is released, the trigger spring 28 expands, returning it andthe piston 26 to a resting position.

Because the piston 26 is sealed within the cylinder 38 by the sealingo-ring 30, its repeated movement causes the air within the cylinder tobe displaced thereby creating a vacuum within the pick-up tube 18 of thewater gun. The composition located in the housing 12 is then drawn intothe pick-up tube 18, past a first check valve 32, past the triggerspring 28, past a second check valve 34, into the mixing chamber 20 andout the nozzle orifice 22 via an outlet tube 24. The second check valve34 is illustrated with a spring mechanism 35 which serves to maintainthe check valve 34 in a closed position isolating the piston assemblycylinders 28 and conduit means 44 from the mixing chamber 20, allowing avacuum to form within the gun during operation.

The same mechanism operates to simultaneously withdraw composition fromthe complementary housing 10 into the mixing chamber 20 via a pick-uptube 16. Thus, referring to FIGS. 2 and 3, the action of the piston 36within its cylinder compresses the trigger spring 43 against the springretainer 42 creating a vacuum within the pick-up tube 16 and moving someof the composition located in the housing 10 through the pick-up tube 16into the mixing chamber 20 and out the nozzle orifice 22.

As illustrated in FIG. 2, the two pick-up tubes 16 and 18 originate inthe housings 10 and 12, respectively. Each pick-up tube 16, 18 includesa check valve 32 and 33, respectively. The first check valves 32, 33serve to prevent fluid flow from the piston assembly cylinders 38, 48back into the housings 10, 12. The single second check valve 34 preventsthe mixed compositions from flowing out of the mixing chamber 20 backinto the piston assembly cylinders 38, 48.

Thus, repeated depression of the trigger 14 increases the pressurewithin the gun, thereby filling the mixing chamber 20 with a combinationof the compositions located in the two housings 10, 12, then forcing themixed compositions through the outlet-tube 24 and out the nozzle orifice22.

Example 2

Dual Chamber Fluid Dispensing Apparatus--Gas-Charged Toy Water Gun

In contrast to the above-described toy water gun, the gas-charged toywater gun operates using pressurized gas, rather than the pistonassembly, to move the bioluminescent mixtures through the system. Apreferred embodiment of this device is illustrated in FIGS. 4 and 5. Inthis embodiment the butt of the water gun 86 houses the two chambers 64,74 that contain the bioluminescence generating system components.Further, the butt 86 is detachable and thus readily replaced.

To pressurize the gun for operation, a CO₂ or air [or other suitable gasor mixtures thereof] canister 50 is inserted into a gas chamber 56 asshown. A screw cap 52, located at the base of the gas chamber, securesthe canister 50 into the chamber 56. As the screw cap 52 is tightened,the CO₂ or air canister is forced against a piercing pin 54, therebyreleasing CO₂ or air into the gas chamber 56 and charging the water gunfor use.

Depression of a trigger 58 aligns a gas cock 60 with each of two gasconduits 62 and 72 and the gas chamber 56. With the gas cock 60so-aligned, CO₂ gas or air enters the gas conduits 62 and 72 and passesinto the two chambers 64 and 74. The pressure of the gas forces some ofeach mixture out of the chambers 64, 74, via composition pick-up tubes66, 76. The composition pick-up tubes 66, 76 are connected to outletconduits 78 and 80 through which the mixtures pass into a mixing chamber68, and are combined. The continued pressure of the CO₂ gas or airforces the combined mixture from the mixing chamber 68 and out a nozzleorifice 70.

The gas conduits 62, 72 and outlet conduits 78, 80 are housed within themain body of the water gun and extend beyond it in the region where thebutt 86 of the gun is attached to the main body. The composition pick-uptubes 66, 76 are completely within the butt of the water gun 86. Inorder to obtain a leak-free assembly of the butt of the gun to the mainbody, the gas conduits 62, 72 and outlet conduits 78, 80 each passthrough a leak seal 88 located within the butt of the gun 86. The leakseals 88 may be constructed of rubber or similar soft sealing materialand should be covered, either with a removable cap or with a materialsusceptible to piercing, to prevent spillage of the compositionscontained therein.

In attaching the butt of the gun 86 to the main body, the gas conduits62, 72 and outlet conduits 78, 80 pass through the leak seals 88 forminga tight seal between the tubes and the butt of the gun. Also, as can beseen in FIG. 4, the delivery tubes 78, 80 set within the compositionpick-up tubes 66, 76 at the point where they enter the butt of the gun.This permits fluid communication between the composition pick-up tubes66, 76 and the outlet conduits 78, 80.

Additional features of the preferred embodiment, as illustrated in FIGS.4 and 5 include retaining hooks or latches 90, 92 and 94 positioned onthe main body of the water gun and used to secure the butt of the gun tothe main body. Additionally, the two chambers 64 and 74 can beconfigured with filler caps 82 and 84, as illustrated, thereby allowingthem to be refilled as an alternative to replacement.

It will be appreciated that the gas used to operate the gas-chargedfluid dispensing apparatus described herein may be other than carbondioxide. Any gas or mixture of gases, such as air or mixtures of O₂ andCO₂, that operates in the same manner may be used.

Example 3

Dual Chamber Fluid Dispensing Apparatus--Gas-Charged

FIGS. 6, 7 and 8 illustrate a preferred embodiment of a gas-chargedfluid dispensing apparatus as provided herein. This embodiment may beadapted for particular uses; for example, it may be housed within adecorative sculpture, thereby functioning as a decorative waterfountain. Alternative embodiments incorporating this embodiment areillustrated in FIGS. 4 and 5 [EXAMPLE 2] and FIGS. 9 and 10 [EXAMPLE 4].

Referring to FIGS. 6 and 7, the gas-charged dual chamber dispensingapparatus has two chambers 100 and 102. In a preferred embodiment asillustrated, the two chambers 100 and 102 are refillable via filler caps104 and 106 located on the upper end of the chambers. A gas chamber 108is situated about equidistant from the two chambers and communicateswith each of them via gas conduits 117. The gas conduits 117 end at gasinlets 118 that communicate with the two chambers 100, 102. The gasinlets 118 are positioned near the upper end of the chambers 100 and102. While one gas inlet 118 is depicted, it is understood that eachchamber 100, 102 has such an inlet.

A gas canister 112 fits into the gas chamber 108, being secured thereinby a screw cap 110. Screwing the screw cap 110 tightly into place forcesthe top of the gas canister 112 against a piercing needle 114, therebyreleasing gas into the gas chamber 108. A gas control valve 116 is usedto control the flow of the gas from the gas chamber 108 into the gasconduits 118.

A mixing chamber 124 is also situated about equidistant from the twochambers 100 and 102 and communicates with them via outlet conduit means122, such as fluid ports. The outlet conduits [fluid ports] 122 arelocated sufficiently near the bottom of the chambers 100 and 102 topermit the chamber contents to empty. Near the lower end of the twochambers 100, 102 are fluid outlets that connect to the fluid ports 122.Blow-out plugs 120 prevent the compositions contained therein fromleaving the chambers and entering the fluid ports before activation ofthe device. One-way valves or similar devices can be substituted for theblow-out plugs 120. The mixing chamber 124, having bottom inlets and atop outlet, is associated with a nozzle 126, which may be attached orintegral to the mixing chamber. Optionally, the nozzle 126 has a closurecap 132 distal to the mixing chamber 124.

In a preferred embodiment, illustrated in FIGS. 6, 7 and 8, an uppersupport 130 is shown. This upper support 130 spans the upper ends ofboth chambers 100 and 102 and over the top end of the gas chamber 108.The gas conduits 118 and inlets 117 are within the upper support 130.The nozzle 126 passes through the upper support 130 and is supportedthereby.

Also illustrated in this preferred embodiment, is a base support 123that spans across the lower ends of the chambers 100 and 102 and that isintegral to the mixing chamber 124. The fluid ports 122 connecting thechambers 100 and 102 with the mixing chamber 124 are contained withinthe base support 123 [see, FIGS. 6 and 7].

To operate the basic dual chamber gas-charged fluid dispensingapparatus, a gas canister 112 containing gas under pressure, for examplepressurized CO₂, is inserted into the gas chamber 108. The screw cap 110is tightened, forcing the gas canister against the piercing needle 114.As gas escapes from the canister, it fills the gas chamber. The gascontrol valve 116 is opened, permitting the gas to enter the gasconduits 117 and pass into the chambers 100 and 102 through the gasinlets 118.

The pressure of the gas in the chambers pushes the mixtures thereinagainst the blow-out plugs 120, or through the one-way valves, out thefluid outlets, into the fluid ports 122 or other fluid conduit means,and into the mixing chamber 124 via the bottom inlets. In the mixingchamber 124, the mixtures combine, while the continued pressure from thegas propels the combined mixtures through the nozzle 126 and out thenozzle orifice 128.

Example 4

Dual Chamber Fluid Dispensing Apparatus and Volcano-Shaped Gas-ChargedApparatus

FIGS. 9 and 10 illustrate a preferred embodiment of the gas-chargedfluid dispensing apparatus illustrated in FIGS. 6, 7 and 8 and describedabove. In this embodiment, each chamber has a generally half-conicalshape, or other suitable shape [depending upon the intended use], suchthat, when attached they form, in this embodiment, a volcano-shapedapparatus. The gas chamber 160 and gas conduit 162 are defined by theinner walls 176, 178 of the chambers 150, 152, respectively. Similarly,the mixing chamber 170 and nozzle 172 are defined by the inner walls176, 178 of the chambers 150, 152, respectively.

As in the apparatus, FIGS. 6, 7 and 8, a gas canister 154 is housed inthe gas chamber 160 and is activated by tightening a gas screw-cap 156which forces the gas canister 154 against a piercing needle 158 therebyreleasing the gas into the gas chamber 160. The gas enters the gasconduits 162, forces out the blow-out plugs 164 and passes into thechambers 150, 152 via the gas inlets 166. Alternatively, a controlvalve, or other suitable control means, is situated between the gaschamber and gas conduits or within the gas conduit means and used tocontrol the flow of gas into the gas chambers.

Within the two chambers 150, 152, one containing, for example, up to allexcept one component necessary for the bioluminescence generatingreaction and the other the remaining component(s), the gas forces thebioluminescence generating mixtures into the mixing chamber 170.Blow-out plugs 168, situated between the chambers 150, 152 and mixingchamber 170, prevent the bioluminescence mixtures from entering themixing chamber 170 until the apparatus is activated. The continuedpressure of the gas forces the combined mixtures from the mixing chamber170 through the nozzle 172 and out the nozzle orifice 174.

This apparatus is particularly designed for use as "fireworks"configured in the shape of a volcano. As the combined bioluminescentmixtures are forced from the apparatus into the air, they glow in asimilar manner to traditional fireworks.

Alternatives to the specific embodiment described herein are likewisecontemplated. For example, blow-out plugs may be replaced by one-way orcontrol valves. Manually operated valves may be replaced byelectronically or mechanically controlled valves. The apparatus does nothave to be in the shape of a volcano, but may be formed into any shape,such as animals, humans, plants or abstract forms.

In another alternative embodiment, not illustrated, the nozzle 172,through which the mixed bioluminescent composition exits from theapparatus, is shortened, moving the mixing chamber 170 closer to thenozzle orifice 174. This is particularly appropriate where thebioluminescence generating system used in the apparatus produces shortbursts of light or is quickly exhausted once activated, such that thebioluminescent components are preferably kept separated until justbefore expulsion from the apparatus. In such an alternative embodiment,outlet tubes (or conduits) may be provided that maintain thebioluminescence generating components separate until just beforeexpulsion from the apparatus. The outlet tubes illustrated in FIGS. 23,24 and 26 and described in EXAMPLE 11, could likewise be employed inthis alternative embodiment.

Example 5

Compressible Dispensing Apparatus--Lotion/Cream container

FIG. 11 illustrates a preferred embodiment of a compressible dispensingapparatus particularly useful for dispensing waxy, pasty or semi-solidcompositions such as body lotions or finger paints. In this embodiment,the container, preferably a tube, has two chambers 200, 202. In certainembodiments, within one chamber are all, except for one or more,components of the bioluminescence generating system, and in the otherchamber are the remaining components. The composition, such as bodylotion or cream is in one or, preferably, both chambers. The containeris preferably constructed of a pliable collapsible or compressiblematerial, such as plastic, plastic/metal laminate or similar collapsiblecomposite, which can be squeezed by hand. Numerous such tubes are knownto those of skill in this art are used to dispense products such asfinger paints, toothpaste, gels, lotions and other such items.

A membrane seal 204 at the top end [dispensing end] of the containerprevents the contents of the chambers from mixing. The cap apparatus 206of the container has a dispensing cap at the top 210 and is configuredsuch that a space 208 exists between the membrane seal 204 and thedispensing cap 210, which space acts as a mixing chamber 208.

Thus, to operate the lotion/cream container, the membrane seal 204 ispunctured, or otherwise opened, and a portion of the contents of the twochambers 200, 202 are simultaneously squeezed into the mixing chamber208 by applying pressure to the container. The dispensing cap 210 isremoved and the contents of the mixing chamber 208 are squeezed out thedispensing orifice 212. The mixed composition may be dispensed bysqueezing the container or by squeezing the cap apparatus 206.Alternatively, a plunger/syringe device [not illustrated] may beattached to the dispensing orifice and the mixed cream compositionthereby withdrawn from the mixing chamber 208.

The membrane seal, 204 situated between the chambers 200, 202 and themixing chamber 208, functions to prevent the contents of the mixingchamber 208 from returning into either of the chambers 200, 202. It maybe constructed, for example, of a thin layer of rubber, plastic, orother suitable porous material, having a small hole or holes throughwhich the contents pass. As the sides of the container are compressed,portions of the contents of the chambers are forced through the holes inthe membrane and into the mixing chamber, with the membrane returning toits "sealed" state once the pressure is relieved. A one-way valve orsimilar device may be substituted for the membrane seal 204, provided ittoo prevents the contents of the mixing chamber 208 from flowing backinto either of the chambers 200, 202.

Example 6

Bottle/Bladder Apparatus--Bubble Composition Bottle

FIGS. 1 2 and 13 illustrate a preferred embodiment of the bottle/bladderapparatus adapted for use with bioluminescent bubble compositions. Thisbubble composition bottle has a bladder 300 positioned within it andheld in place, in the neck 302 of the bottle, by friction. A collar 304is positioned on the neck of the bottle 302, preventing the cap 306 frombeing screwed completely onto the top of the bottle. The cap 306contains a plunger 308 which operates to push the bladder 300 into thebody of the bottle when the collar 304 is removed and the cap 306 isscrewed down tightly. Upon entering the body of the bottle, the bladderis pierced by a piercing pin 310 located on the bottom of the bottle;thereby releasing the contents of the bladder into the bottle. FIG. 13shows the bottle with the collar 304 removed, the cap 306 screwed ontightly, and the bladder 300 collapsed within it.

Component(s) [less than all] of the bioluminescence generating reactionare contained in the bladder. The components may be in the form of asolution, suspension, suspended particles, or particles. Prior to usethe bottle may be gently agitated. The particles may be time releasecapsules that release their contents upon exposure to the bubblecomposition or from which the contents diffuse upon mixing of thecontents of the bladder with the bubble composition. The remainingcomponent(s), such as Ca²⁺ or ATP, are contained in the bubblecomposition 314, which is preferably a mild bubble forming composition.Selection of the bioluminescence generating composition depends upon theselected bubble composition and also the desired action. In otherembodiments, remaining components, such as ATP, FMN, a flavin reductaseor other component that may be somewhat sensitive to the bubblecomposition, of the bioluminescence generating system may be added tothe bubble composition just prior to use.

The collar 304 of the bottle is adapted with a bubble blowing ring 312,with arm, integral thereto. Thus, the collar 304 is removed, the bladder300 pierced within the bottle as described and the bubble blowing ring312 dipped into the mixed composition, withdrawn and bioluminescentbubbles blown. A standard bubble blowing wand [arm with ring] may beused and/or provided in place of that depicted in FIG. 12.

The bladder 300 should be constructed of a material that can be piercedby a piercing means, such as a needle or pin, made for example of thinplastic or other polymeric film. Preferably the distance from the baseof the neck of the bottle to the tip of the piercing needle is less thanthe length of the bladder, so that the bladder will be pierced by theneedle before its top edge clears the base of the neck of the bottle.

The bottle 316 may be fabricated of any material ordinarily used fordispensing bubbles. It may be transparent or translucent to thebioluminescent light so that any glow in the bottle can be seen.

Example 7

Container/Bladder Apparatus--Beverage Can

An exemplary of the container/bladder apparatus, illustrated in FIG. 14,is suitable for use as a beverage can or bottle. It is configuredsimilarly to a pop-top aluminum drink can but has a bladder 400 underthe top which is pierced by the pop-top 402 when the can is opened. Thebladder may be centered under the top of the can, as illustrated, may beoff-center or may be attached to the top and side of the can.Positioning of the bladder is chosen such that it may be readily piercedand its contents mixed with the contents of the container 404. Thus, thebladder should be sufficiently thin that the pop-top 402 is able topierce it allowing its contents to mix with the contents of the beveragecan. The can is preferably fabricated of translucent or transparentmaterial such that the glowing beverage can be observed.

An alternative embodiment includes a beverage container with twopop-tops, in which one is designed, such as including by having a pointat the end, to puncture the bladder and the other can be a typicalpop-top that is used for emptying the contents of the can, such as bypouring into a glass or into a person's mouth. Since the novelty ofthese items resides in the resulting glow in the beverage, the beverageshould be poured into a glass, or the container should be transparent ortranslucent to the bioluminescent light.

Another alternative contemplated herein includes a mesh filtersurrounding the bladder and functioning to prevent small pieces of theruptured bladder from mixing with the contents of the can. The contentsof the bladder are in aqueous composition; thus, the density of the meshof the filter that is permeable to the luciferase and otherbioluminescence generating components.

Similarly, embodiments employing other opening types are contemplatedherein. For example, the bladder and corresponding container opening maybe pierced with a point-ended straw, or other sharp device. Likewise,the dispensing opening [which may be the same as the bladder-associatedopening] may be covered with a thin aluminum pull tab. Critical to theoperation of the can/bladder combination is that the bladder precludemixing of the contents of the bladder and the can until the consumertakes action to rupture the bladder.

The bladder may be constructed of any material which is amenable tobeing pierced as described and is preferably constructed of a materialwhich will rarely if ever break into small pieces when pierced. Forexample, aluminum foil with a thin plastic coating, when pierced with apoint-ended straw in particular, will rarely break into small pieces.The body of the can may be constructed of aluminum, plastic or similarmaterial and is preferably constructed of a translucent material such asplastic.

The bladder includes up to all except for one component of thebioluminescence generating system, and the beverage includes theremaining component(s). For example, the bladder includes the aequorinphotoprotein [typically 0.1 to 1 mg or more] in a composition containinga chelator to prevent activation of the photoprotein, and the beveragecontains Ca²⁺.

Example 8

Single Use, Dual Chamber Fluid Packaging Apparatus

FIG. 15 illustrates an exemplary embodiment of the single use, dualchamber fluid packaging apparatus or bottle described generally above,and the following description is with reference to that FIGURE. Thebottle has a first chamber 500 which contains a composition includingone or more, up to all but one, of the bioluminescence generating systemcomponents. Below the first chamber and operatively attached thereto, isa second chamber 502, containing the remaining bioluminescencegenerating system components in composition. In the embodimentillustrated, the first chamber 500 is seated in the second chamber 502along a side seam 506 and a separation membrane 504.

The second chamber 502 is constructed of pliable material, such asplastic, that is convoluted 508 such that it can be readily collapsedagainst the bottom of the first chamber in the direction of theillustrated arrow. When collapsed in this way, the force of thecomposition contained within the second chamber ruptures the separationmembrane 504A, permitting the compositions to mix. Once mixed, thecompositions begin to illuminate.

This apparatus, as illustrated, is adapted for use with bubble-blowingcompositions in that the cap of the bottle 510 has a bubble-blowing wand512 attached to it. Alternatively, the apparatus may be used with abeverage and, if so used, would not have the illustrated bubble-blowingwand 512.

Another embodiment of this apparatus, not illustrated, but contemplatedherein, is a bottle in which the second chamber may be secured to thefirst chamber or to itself in a collapsed position. For example, thesecond chamber can be adapted with a hooking mechanism on its exteriorsuch that it can be hooked to itself when collapsed.

Example 9

Cap Apparatus for Use with Composition Vessels

FIGS. 16, 17 and 18 & 19 illustrate three exemplary embodiments of thecap apparatus for use with composition vessels.

A. Cork Cap Apparatus

Referring to FIG. 16, a cork 600, situated within the neck 602 of abottle and having a rupturable capsule 604 housed within it, isillustrated. In this embodiment, the bottom edge of the cork 600 issubstantially U-shaped such that a pocket is formed. Contained withinthe pocket is the capsule which is in communication with the screen 608which is permanently attached to the bottom of the cork. The capsulecontains one or more, up to all but one, of the bioluminescencegenerating system components. A plunger assembly 606 is positioned,partially within the cork, such that depression of the plunger assembly606 results in rupture of the capsule and release of its contents intothe composition within the bottle. The screen 608 or other filteringdevice prevents fragments of the ruptured capsule from entering thevessel.

The plunger assembly 606, illustrated in FIG. 16, has a top portion 610integral to the stem portion 612. Pressing on the top portion 610 forcesthe stem 612 to move within the cork 600 and against the capsule 604,thereby rupturing the capsule and releasing its contents into thevessel.

FIG. 17 illustrates an alternative embodiment of the cork cap apparatus.In this embodiment, the cork 700 is illustrated as being about flushwith the top of the neck 702 of the bottle. The plunger apparatus 704 isadapted with a finger ring 706 for ease in handling. The stem 708, whichmay be pointed or blunt or any combination thereof, is threaded 710. Inoperation, the plunger assembly 704 is screwed into the cork 700 whereit contacts a capsule 712, rupturing it and releasing its contentsagainst the screen 714 or filter. The capsule will preferably containpowdered or otherwise condensed bioluminescence generating components.

It will be appreciated that the cork cap alone, with encapsulatedcompositions encased within and screen or filter attached thereto, is analternative embodiment of the two illustrated cork cap apparatus. Inthis embodiment a corkscrew may be employed to rupture the capsule andto remove the cork cap.

B. Screw-top Cap Apparatus

FIGS. 18 and 19 illustrate another exemplary embodiment of the capapparatus for use with composition vessels. FIG. 18 shows the capapparatus before activation or engagement. This is particularly adaptedfor use with a wine or champagne bottle, and includes encapsulatedbioluminescence generating system components.

This embodiment generally includes a bottle-shaped vessel with a collar802 situated about the neck 804 of the bottle and a cap 800 attached tothe top of the bottle just above the collar 802. The neck of the bottle804 is threaded to receive the screw-on cap 800. The collar 802 issituated such that a lower portion of the threads on the neck of thebottle 804 are covered thereby preventing the screw-on cap 800 frombeing completely attached to the bottle. Enough threads remain exposedon the top of the bottle such that the screw-on cap 800 is securely,though not completely, attached to the top of the bottle.

The screw-on cap 800 has a plunger 806 integral thereto which extendsinto the bottle neck 804. A screen or filter assembly 812 is attached tothe interior of the bottle within the bottle neck 804. A membrane system808, 810 or capsule or similar composition packaging is situated betweenthe plunger 806 of the screw-on cap 800 and the screen/filter assembly812. In operation, the collar 802 is removed, for example by removingthe screw-cap 800 and lifting off or screwing off the collar 802 or bytearing off the collar 802, and the screw-on cap 800 is tightenedagainst the top of the bottle. This forces the plunger 806 through themembranes 808, 810, rupturing them and releasing the compositionscontained therein. The composition(s) pass through the screen assembly812 and are mixed with the contents of the bottle. FIG. 19 illustratesthe cap apparatus fully engaged with the membrane system ruptured.

In the embodiment illustrated, the screen assembly 812 is attached alongthe interior of the neck of the bottle 804 as well as across theinterior of the neck, thereby forming a basket within which the membranesystem 808, 810 sits. Alternatively, the screen assembly can be attachedaround the circumference of the bottle neck only and not along its sidesto the top of the bottle, as illustrated.

The precise height of the collar 802 will be determined by the length ofthe plunger 806 and location of the membrane system 808, 810. The heightwill be sufficient to prevent the plunger 806 from being engaged throughthe membrane system 808, 810 prior to activation by the user, whilepermitting the screw-on cap 800 to be secured to the top of the bottle.

The membrane system 808, 812 contains one or more, up to all but one, ofthe bioluminescence generating system components. Typically thecomponents will include the luciferase and luciferin in lyophilizedform.

The illustrated embodiment is shown and described as attached to abottle. It will be appreciated, however, that the vessel to which thecap apparatus is attached may be a can, tube or any other container.Additionally, the embodiment is exemplified and illustrated withreference to the neck of the bottle. It is not necessary that the vesselhave a "neck" for the cap apparatus to function. For example, if thevessel does not have a neck, other means may be employed to hold thecollar in place below the screw-on cap, such as, a lip formed on thecontainer, below the threads, to stop the collar at an appropriatepoint.

With respect to these three embodiments of the cap apparatus adapted foruse with composition vessels, the stem of the plunger assembly is shortenough not to pierce the screen or filter device, yet long enough toeffectively rupture the capsule, membrane or other packaging onceengaged. The bioluminescence generating system component(s) containedwithin the cap apparatus may be powdered or in composition or in anyform amenable to addition to the composition contained within thevessel. Additionally, the components may be contained in more than onecapsule, membrane or other packaging. In this case, the componentpackages are adjacently positioned, such that each is ruptured byengagement of the plunger. Preferably, the remaining components requiredfor completion of the bioluminescent reaction are contained within thevessel within any composition. These embodiments are particularlyadapted to use with wine or champagne or other beverage.

Example 10

Spray container apparatus

FIGS. 20, 21 and 22 illustrate an exemplary embodiment of a spraycontainer provided herein. This container is typically a can apparatusintended for use in combination with the bioluminescence generatingsystems as described herein. The following description of that exemplaryembodiment is made with reference to those figures.

The spray container apparatus includes two portions, a top housingportion 902 and a bottom plunger portion 904. The contents of the tophousing portion 902 include all, except one or more, of the componentsof a bioluminescence generating system. The top housing portion 902 alsocontains a conduit 912 operatively attached to a spray nozzle 920.

The top housing portion 902 of the spray container apparatus is adaptedto receive the bottom plunger portion 904. In this embodiment, the tophousing portion 902 and bottom plunger portion are threaded 903 and 910,respectively, such that the bottom plunger portion 904 can be screwedonto the top housing portion 902. [See FIG. 21, illustrating the spraycontainer apparatus with the bottom plunger portion fully screwed intoplace.]

The top housing portion 902 additionally has a pocket 926 defined by aconical side wall 922 and a top wall/rupture membrane 916. The pocket926 is adapted to receive a pellet 906, that contains the remainingcomponent(s) necessary for generating bioluminescence.

The bottom plunger portion 904 of the spray container apparatus has aplunger 914 shaped and situated such that it fits into the pocket 926 ofthe top housing portion 902 when the bottom plunger portion 904 isscrewed tightly in place. The bottom plunger portion 904 is adapted withan angular seal 918 that serves to seal the bottom plunger portion 904against the top housing portion 902 thereby preventing leakage of thecontents of the spray container apparatus.

In operation, the pellet 906 is placed into the pocket 926 of the tophousing portion 902 where it contacts the top wall/rupture membrane 916of the pocket 926. The bottom plunger portion 904 is then screwed ontothe top housing portion 902, thereby forcing the plunger 914 against thepellet 906, which presses against the top wall/rupture membrane 916 ofthe pocket 926, rupturing the same. The pellet dissolves or is suspendedin the composition contained in the top housing portion 902 and thecomposition glows. Depression of the spray nozzle 920 releases thecontents of the spray container apparatus.

Alternative embodiments of this spray container apparatus will beappreciated. For example, the pellet 906 may be a vessel containing thenecessary bioluminescence generating components that is fabricated frommaterial that can dissolve or that will be suspended in the compositioncontained in the top housing portion 902 of the spray containerapparatus 900 or that will release its contents upon contacting thecomposition, such as by passive diffusion. Examples of such materialinclude, but are not limited to liposomes, gelatin, soluble paper andother such materials that will dissolve or release contents into aqueouscompositions. Further, the spray container apparatus 900 can be adaptedsuch that the bottom plunger portion 904 snaps onto the top housingportion 902, rather than screwing into place.

Example 11

Alternative Embodiment of Dual Chamber Fluid Dispensing Apparatus--ToyWater Gun

Another embodiment of the dual chamber fluid dispensing apparatus is atoy water gun, such as that illustrated in FIGS. 23 through 26. This toywater gun includes two housings [or chambers] 406, 408 that may beconstructed of injection-molded plastic or other suitable material. Thetwo housings 406, 408 are affixed, such as glued, heat sealed or byother such means, along a median seam 462 to form the body of the watergun. See especially FIGS. 25 and 26.

In operation, one housing 406 contains a mixture having less than allthe components necessary for generating bioluminescence and the otherhousing 408 contains a mixture having the remaining components or theremaining components except for air. Depressing the trigger 410 pushesthe pistons 428, 430 into their respective cylinders 450, 452compressing the trigger springs 432, 434 and pushing the contents of thecylinder through the respective conduit means 458, 460, past the secondcheck-valves 442, 444, out the outlet tubes 424, 426, into the mixingchamber 420 and out the nozzle orifice 422. As the trigger 410 isreleased, the trigger springs 434, 432 return to their relaxed statepushing the pistons 430, 428 out of the cylinders 452, 450 creating avacuum therein that pulls the contents of the housings 406, 408 throughthe pick-up tubes 412, 414, past the first check-valves 438, 440 andinto the cylinders 450, 452. Pumping the trigger, such as by repeatedlydepressing and releasing it, moves the mixtures contained in thehousings through the gun into the mixing chamber 420 and out the nozzleorifice 422.

As the mixtures leave the outlet tubes 424, 426, just prior to expulsionfrom the toy gun via the nozzle orifice 422, they enter the mixingchamber 420. Bioluminescence begins either upon mixing of the componentsor as the mixed composition contacts the air as it exits the toy gun.The mixtures may be powdered, such as those produced by lyophilization,or they may be condensed into a paste, or they may be liquid. Ifpowdered or condensed, water or a suitable composition, such as asuitable buffer can be added prior to use.

The housings 406, 408 may be filled and refilled through the fillingcaps 464, 466 located at the top of each housing. The trigger 410 isattached to a trigger guide 416 which serves to guide the trigger 410towards the two piston assemblies 472. Only one of the two pistonassemblies 472 is completely illustrated, and it is visible in FIG. 23.The other piston assembly is adjacent to and, in this embodiment,identical to the one illustrated. Depression of the trigger 410activates the two piston assemblies, e.g., 472. This causes a portion ofthe composition located in each housing 406, 408 to move through the toygun into a mixing chamber 420 and out a nozzle orifice 422, as detailedabove.

The piston assemblies e.g., 472 each include a piston 430, 428 whichpasses through a sealing o-ring 436, 429 is connected to a triggerspring 434, 432 and moves within a cylinder 452, 450. The pistonassemblies each also include a spring retainer 456, 454 that secures oneend of the trigger spring 434, 432 to the end wall of the cylinder. Eachcylinder 452, 450 is in communication with one end of a pick-up tube414, 412 and is about perpendicular to the pick-up tubes 414, 412. Eachcylinder 452, 450 also communicates with the conduit means 458, 460.

Because the pistons 428, 430 are sealed within their cylinders 450, 452by a sealing o-ring 429, 436, repeated movement of the pistons withinthe cylinders causes the air within the cylinders to be displacedthereby creating a vacuum within the pick-up tubes 412, 414 of the toygun. This initiates the operation of the toy gun as described in detailabove.

The illustrated embodiment has a trigger guard 411 that acts to preventaccidental discharge of the gun and makes the gun appear more realistic.The sighting aids 468, 470 aid in aiming the toy gun and also serve tomake the gun appear realistic.

As illustrated in FIG. 25, the two pick-up tubes 412 and 414 originatein the housings 406 and 408, respectively. Each pick-up tube 412, 414includes a check-valve 440, 438, respectively. The first check-valves440, 438 serve to prevent fluid flow from the piston assembly cylinders450, 452 back into the housings 406, 408. The second check-valves 442,444, similarly prevent the fluids from flowing out of the outlet tubes424, 426 and back into the piston assembly cylinders 452, 450.

Thus, in operation, repeated depression of the trigger 410 increases thepressure within the gun, thereby filling the mixing chamber 420 with acombination of the compositions located in the two housings 406, 408,then forcing the mixed compositions out of the toy gun through thenozzle orifice 422.

Example 12

Compressible Dispensing Apparatus

FIG. 27 illustrates an alternative exemplary embodiment of acompressible dispensing apparatus. This embodiment is particularlyadapted for containing and dispensing bioluminescent slimy play materialas described herein, but may be used to dispense other ingredients. Theprimary difference between the embodiment illustrated in FIG. 11 andthat illustrated in FIG. 27 is that the latter has one or more smallcompartments 942, 944 located within the apparatus. These compartmentsare located such that compression of the apparatus expels the contentsof the compartments into the main body 940 of the apparatus where thosecontents and any contents contained within the main body 940 mix.

The embodiment illustrated in FIG. 27 has a first compartment 942 and asecond compartment 944 contained within the main body 940 of thecompressible dispensing apparatus. The compartments 942, 944 arepreferably formed, along at least one edge 950, 952, by rupturablemembranes, such as plastic membranes, or other readily punctureddividing means. At least one other edge of each compartment 946, 948 ispermanently affixed to the interior of the main body 940 of theapparatus. Thus, upon compression of the apparatus, the contents of thetwo compartments 942, 944 press against and rupture the rupturablemembranes 950, 952, resulting in expulsion of the contents of the twocompartments 942, 944 into the main body 940 of the apparatus. Becauseat least one edge of each compartment 946, 948 is permanently affixed tothe interior of the apparatus, the compartments remain in position andreadily rupture during compression.

Preferably the two compartments 942, 944 are large enough to contact oneanother along one contact edge 954 within the apparatus. As the sides ofthe apparatus are compressed, the contents of the two compartments arepressed against this contact edge 954 and against the rupturablemembranes 950, 952, which membranes then rupture. Preferably, the cap956 to the apparatus remains in place until the two compartments havebeen ruptured and the contents mixed within the apparatus.

The compressible dispensing apparatus is illustrated in FIG. 27 with twocompartments 942, 944; however, it will be appreciated that one, threeor more compartments may be included as appropriate. Factors to beconsidered in determining the appropriate number of compartments are thebioluminescence generating system to be used, the ingredients,particularly slimy play material ingredients to be used, the desiredtiming and duration of illumination, and the ultimate use for resultingcomposition, such as the slimy play material.

By way-of example only, where two compartments are included in theapparatus, as illustrated in FIG. 27, one compartment may contain thecharged luciferin/luciferase mixture, such as aequorin photoprotein withcoelenterazine and oxygen and the second compartment may contain apolyvinyl alcohol mixture. The main body of the apparatus contains theremaining ingredients, such calcium ions, necessary to complete thebioluminescence generating reaction, and also contains the otheringredients of the slimy play material, such as sodium tetraborate.

Alternatively, where the apparatus is configured with three compartmentswithin the main body, one or more of the ingredients contained withinthe main body of the two compartment embodiment may instead be containedwithin the third compartment. For example, the sodium tetraborate may beincluded in the third compartment and the calcium ions, in an aqueousmedium, may be in the main body of the apparatus. It will further beappreciated that the contents of each compartment and/or the main bodymay be in powder, liquid or semi-solid form. The liquid or semi-solidform are preferred.

Example 13

Recombinant production Renilla reniformis luciferase

The phagemid pTZ18R (Pharmacia) is a multi-purpose DNA vector designedfor in vitro transcriptions and useful for expression of recombinantproteins in bacterial hosts. The vector contains the β-lactamase gene,which allows for the selection of transformants by resistance toampicillin, and a polylinker site adjacent to the lacZ' gene. Theheterologous gene of interest is inserted in the polylinker andtranscribed from the lac promoter by induction, for example, withisopropyl-β-D-thiogalactopyranoside (IPTG).

The DNA encoding the Renilla reniformis luciferase has been cloned(e.g., see U.S. Pat. Nos. 5,292,658 and 5,418,155). The plasmidpTZRLuc-1 encodes the Renilla luciferase on a 2.2 Kbp EcoRI to Sstl DNAfragment inserted in EcoRI and Sstl sites of pTZ18R (plasmidconstruction is described U.S. Pat. Nos. 5,292,658 and 5,418,155; seealso Lorenz et al. (1991) Isolation and Expression of a cDNA encodingRenilla reniformis Luciferase, Proc. Nati. Acad. Sci. U.S.A.88:4438-4442). The initiation of transcription of the Renilla luciferasecDNA is under the control of the lacZ' promoter. E. coli strainsharboring plasmid pTZRLuc-1 express Renilla luciferase that isfunctional in bioluminescence assays and retains the most of thecritical properties of the native enzyme (see, e.g., U.S. Pat. Nos.5,292,658 and 5,418,155).

A derivative of pTZRLuc-1, pTZRLuc-3.6, produces approximately 7-foldhigher levels of recombinant Renilla luciferase than pTZRLuc-1 whentransformed into the same E. coli host. Competent E. coli strain XL-1was transformed using purified pTZRLuc-3.6 according to the instructionsprovided by the manufacturer (XL-1 Supercompetent cells™ and protocol;Stratagene, Inc., La Jolla, Calif.). Transfectants were selected byplating on Luria Broth (LB) plates supplemented with 100 μg/mlampicillin.

Single ampicillin resistant colonies were grown in LB mediumsupplemented with 100 μg/ml ampicillin at ambient temperature usingcontinuous shaking until cell growth reached mid-log phase (i.e., cellculture reaches an O.D._(600nm) =0.6-0.8 units). Transcription from thelac promoter was induced by addition of 1 mM IPTG and cell culture wasshaken at ambient temperature for an additional 8 hours.

Cells were harvested by centrifugation at 10,000 x g and frozen at -20°C. The cell pellet was thawed and resuspended at a 1:5 ratio (w/w) in acompositions containing 10 mM EDTA, pH 8.0, containing 4 mg/ml lysozyme(Sigma Chemical Corp.). The cells were placed in a 25° C. water bath for30 minutes and then transferred to ice for 1 hour. The cells were lysedby sonication at 0° C. using a 1 minute pulse from an Ultrasonics, Inc.cell disruptor.

The lysed cellular debris was removed by centrifugation at 30,000 x gfor 3 hours and the supernatant was decanted and retained. The pelletwas resuspended at a 1:5 ratio in the above-described compositions, andthe subsequent incubations, lysis and centrifugation steps wererepeated. The two supernatants were combined and stored at -70° C.

The resulting "clarified lysate" was employed as a source of recombinantluciferase. Alternatively, the lysate may be subjected to additionalpurification steps (e.g., ion exchange chromatography or immunoaffinitychromatography) to further enrich the lysate or provide a homogeneoussource of the purified enzyme (see e.g., U.S. Pat. Nos. 5,292,658 and5,418,155).

Example 14

Cartridges for loading, charging, recharging and/or fillingbioluminescent novelty items

An exemplary loading, recharging or charging cartridge is depicted inFIGS. 28-34. Referring first FIG. 28, a charging cartridge is shown andgenerally designated 1000. This charging cartridge includes a block 1002having two cylinders, a first cylinder 1010 and a second cylinder 1012,and a plunger 1004 having a first piston 1006 and a second piston 1008.Additional chambers may be included. Also, the device may be adapted foruse with the single chamber apparatus provided herein.

As shown, the block is formed with two cylinders 1010 and 1012, and theplunger is formed with two cylindrical pistons 1006 and 1008. It is tobe appreciated that a triangular, rectangular, or any other geometryvessel may be substituted for either cylinder, so long as the shape ofthe pistons provides for insertion into the block. Additionally, forexample, the plunger 1004 may be formed such that the two pistons 1006and 1008 are separate from the other to permit the insertion of pistons1006 and 1008 into the block 1002 at different times.

The block 1002 and plunger 1004 may be made of any material known to oneof skill in the art that does not react with the components of abioluminescence generating system. In a preferred embodiment, the block1002 and plunger 1004 are made of a plastic material that can be readilyinjection molded into a selected particular shape. Suitable plasticsinclude, but are not limited to polyvinyl chloride (PVC), or any otherplastic, TEFLON, polyethylene, or any other material that is inert tocomponents stored and dispensed from the block 1002. Alternatively, theblock 1002 and plunger 1004 can be made from a metal that is machined,cast, or otherwise formed into the particular shape.

Referring now to FIG. 29, the first cylinder 1010 has a plug 1016 whichretains, for example, dry ingredients 1018 containing one or morecomponents of a bioluminescence generating system, preferably includinga luciferase and/or luciferin and any necessary buffers and activators,e.g., ATP or Ca²⁺, and more preferably a luciferase, buffers and anynecessary activators, in lyophilized or other suitable form, in thecylinder 1010 and against the seal 1022. Thus, the dry or condensedingredients 1018 are trapped within the first cylinder 1010 between theplug 1016 and the seal 1022 until the plunger 1004 and piston 1006 areforced into the first cylinder 1010. At that time, theses ingredients1018 are forced through the funnel means 1020, thereby breaking the seal1022, and forcing the ingredients 1018 out of the block 1002 throughnozzle 1024 and out aperture 1026. The seal 1022 is preferably made of amaterial which is capable of being broken with only minimal pressureasserted on the plunger 1004. Such a material includes, for example, apaper, wax-covered paper, plastic sheet, foil, cellophane or any othermaterial exhibiting the requisite properties.

The second cylinder 1012 is formed within a fluid sleeve 1014 that isinserted into the block 1002. In this way, the sleeve 1014 may be asealed tube made from, for example, plastic, glass, or any othermaterial that is compressible and/or breakable, thereby allowing thefluid 1030 to be forced from the sleeve 1014. The sleeve 1014 may beprefabricated and loaded with the fluid 1030 prior to insertion into theblock 1002, or the fluid 1030 may be added to the sleeve 1014 once it ispositioned within the block 1002, and retained therein by plug 1028.

The piston 1008 slides into the second cylinder 1012 and strikes plug1028, advancing it into the block 1002. The advancing plug 1028 createsa fluid pressure within the sleeve 1014 which eventually breaks seal1032 and optionally bathes the matrix material 1034 in fluid 1030. Likethe seal 1022 in the first cylinder 1010, the seal 1032 in the secondcylinder 1014 can be made of any material that can be broken or torn orruptured with only minimal pressure being asserted on the plunger 1008.Such a material may be a paper, wax-covered paper, plastic sheet, foil,cellophane or any other material which exhibits the necessarycharacteristics.

The matrix material 1034 may be any porous material to which thebioluminescence generating component can be adsorbed, absorbed orotherwise linked, as described herein, that is non-reactive with thecomponents of the bioluminescence generating system. When necessary, thematrix material 1034 is included and bathed in the fluid 1030 such thatthe component(s) of the bioluminescnce generating system affixed to thematrix material are released into the fluid 1030. As the piston iscontinually advanced, the fluid, containing bioluminescence generatingcomponents eluted from the matrix material, is forced through the filter1036 and out the nozzle 1038 and aperture 1040. Filter 1036 is used toprevent the expulsion of matrix material 1034 from the second cylinder1014. As a result, the filter 1036 may be made from a cloth or metallicweave, or any other material that will not react with the variouscomponents and compositions present within the second cylinder 1014.

It is to be appreciated, however, that the various components of thebioluminescent reaction may be distributed in different combinationsbetween the two cylinders 1010, 1012, and the matrix material 1034. Onecylinder, such as the first cylinder 1010, typically contains the dry orcondensed ingredients 1018 and the second cylinder 1012 typicallycontains a fluid 1030 and the matrix material containing the remainingcomponents necessary for the bioluminescent reaction. The dry orcondensed ingredients may contain any combination of the components ofthe bioluminescence generating system, such as a luciferase and/or aluciferin, buffer salts, ATP, Ca²⁺ or any other necessary activator. Thefluid 1030 may be water, a buffer, an organic solvent or any otheraqueous medium suitable for solubilizing or suspending one or morecomponents of a bioluminescence generating system to be dispensed intothe bioluminescent novelty item.

In a preferred embodiment, the dry ingredients 1018 include lyophilizedluciferase and buffer salts in powder form, and the fluid includes analcohol that is used to dissolve or suspend a quantity of luciferinaffixed to the matrix material. Alternatively, all of the components ofa bioluminescence generating system, such as the Vargula system, may beadded and packaged in the first and/or second cylinders in the absenceof molecular oxygen such that components are activated when combined andexposed to air.

Referring now to FIG. 30, the cartridge 1000 is shown as used inconjunction with a typical bioluminescent novelty item 1042. As shown,the plunger 1004 has been pressed completely against the block 1002causing the first piston 1006 and the second piston 1008 to be insertedcompletely into the block 1002. As the piston 1006 is advanced into theblock 1002, the dry or condensed ingredients 1018, for example, areforced out of the first cylinder 1010, through the funnel 1020 therebybreaking the seal 1022, and out the nozzle 1024 and aperture 1026 intothe chamber 1044 in novelty item 1042. Likewise, as the piston 1008 isadvanced into the block 1002, the seal 1032 on the sleeve 1014 isruptured causing the fluid 1030 to be dispensed, optionally bathingmatrix material 1034. As the piston 1008 is advanced further, the fluid1030 is forced through filter 1036, out nozzle 1038 and aperture 1040,and into chamber 1046 of novelty item 1042. In this manner, the noveltyitem is fully recharged with the components of a bioluminescencegenerating system necessary for a bioluminescent reaction, whilemaintaining the separation of the chemicals as required for some noveltyitems.

The cartridge 1000 is shown inserted into the filler holes of a typicalnovelty item 1042, such as those described elsewhere in thisapplication. For example, the cartridge could be adapted to fit thenumerous of the novelty items, such as the following novelty items: thefiller caps 17, 19 associated with chambers 10, 12 shown in FIGS. 1 and3; the filler caps 82, 84 shown in FIGS. 4 and 5; the filler caps 104,106 shown in FIGS. 6, 7, and 8; and the filler caps 406, 408 on housing466 in FIGS. 23 through 26. It should be appreciated that althoughseveral novelty items have be identified as being either chargeable orrechargeable using the cartridges disclosed herein, such identificationis merely exemplary and is in no way to be intended as limiting theapplication of the cartridges to those particular novelty items. On thecontrary, the cartridges described herein may be adaptable to charge, orrecharge, virtually any bioluminescent novelty item.

Referring now to FIG. 31, a second embodiment of a charging cartridge isshown and generally designated 1100. The cartridge 1100 is shapedsubstantially like the cartridge 1000, with the addition of a safetyfeature that prevents the accidental or inadvertent discharge of thecartridge when not inserted properly within a novelty item. While anaccidental discharge would not be physically harmful to a human ornon-human animal, such a discharge could prematurely release thebioluminescent materials. The likelihood of such an accidental dischargecould, perhaps, be increased when considering the intended user of manyof the novelty items, such as children.

In this exemplary embodiment, cartridge 1100 contains a block 1102 and aplunger 1104 which, like the cartridge 1000, has a first piston 1106 anda second piston 1108. Unlike the cartridge 1000, however, each of thepistons 1106 and 1108 is equipped with a piston head 1110 and 1112,respectively. These piston heads, in conjunction with cap 1118 preventthe removal of the plunger 1104 from the block 1102. As a result, thecartridge 1100 cannot be disassembled to yield direct access to thecontents of the cylinders 1114 and 1116. In addition to the piston heads1110, 1112, the cartridge 1100 is also equipped with a stop 1120 and aslide 1122 to prevent the accidental compression of the plunger 1104into the block 1102 while the cartridge is not inserted into a noveltyitem. More specifically, the stop 1120 is normally positioned in thepath of the first piston 1106 to prevent the advance of the first piston1106 into the block 1102. Once the cartridge 1100 is positioned on anappropriate novelty item, the slide 1122 is automatically pressedupwards thereby moving the stop 1120 out of the path of the dry piston1106. Once the stop 1120 is out of the way, the two pistons 1106, and1108, may be pressed into the block 1102, thereby releasing the contentsof the first cylinder 1114 and the second cylinder 1116 in the samemanner as discussed above in conjunction with FIGS. 28 through 30.

Referring now to FIG. 32, the cartridge 1100 is shown as used inconjunction with a properly equipped novelty item 1152. As shown, thenovelty item 1152 is equipped with a pin 1162 which extends upwards fromthe novelty item 1152. As the cartridge 1100 is placed over the noveltyitem 1152, the pin 1162 forces the slide 1122 upwards thereby moving thestop 1120 from the path of piston 1106. Once piston 1106 is able to bepressed into the block, the piston 1106 and piston 1108 are forced intothe block 1102. More specifically, as piston 1106 is forced into theblock 1102, the piston advances plug 1126 which in turn forces the dryor condensed ingredients 1128 to break seal 1130. Once the seal 1130 isbroken, the dry or condensed ingredients 1128 are further forced throughnozzle 1132 and out aperture 1134, and into the first chamber 1154 ofthe novelty item 1152. Similarly, as the plunger is depressed, the wetpiston 1108 is forced into the fluid cylinder 1116 and strikes plug1138. As the wet piston is advanced, the plug 1138 creates a fluidpressure within the sleeve 1136, thereby rupturing the seal 1142 causingthe fluid 1140 to be forced through the matrix material 1144, throughfilter 1146, and through nozzle 1148 and out aperture 1150 and into thesecond chamber 1156 in novelty item 1152.

FIG. 33 provides a cross-sectional view of the cartridge 1100, showingin detail the placement of the stop 1120 and slide 1122 in relation tothe dry piston head 1110. As shown, the stop 1120 extends into cylinder1114 sufficiently to prevent the advancement of piston 1126 in cylinder1114. It should be appreciated that while the stop 1120 is blocking theadvance of only the piston 1110, that piston 1112 could be held in placein addition to, or instead of, piston 1110. Moreover, the stop 1120 andslide 1122 could be positioned anywhere in the block 1102 such that thepin 1162 could be positioned on the novelty device in an alternativelocation. It should also be appreciated that a spring (not shown) may beused to hold the stop 1120 in a resting position such that only with themovement of the slide 1122 can the dry piston 1106 be advanced into theblock. Additionally, a spring (not shown) may be positioned to naturallyurge the slide towards hole 1124 in block 1102, thereby preventing theaccidental movement of the slide without the aid of a pin 1162.

In addition to the cartridges as shown above, other means may beemployed to minimize the leakage of the contents of the bioluminescencegenerating systems in combination with the various novelty itemsdescribed herein. More specifically, the novelty item 1152 may beequipped with a removable cap 1164 that is used to seal the chambers1154 and 1156 of the novelty item 1152 to minimize the leakage of anycomponents of the bioluminescence generating system. Further, a seriesof seals 1158 and 1160, or one way seal valves, can be used to preventthe escape of the components once they have been placed in the chambersof the novelty item 1152. Seal 1160 is of a type which is normallybiased to a closed position to prevent the passage of material in onedirection. In this application, the seal 1160 is biased closed such thatany material within the chambers 1154 and 1156 is retained within thechamber. Only upon the insertion of nozzles 1132 and 1148 through theseals 1158 and 1160 is it possible for material to pass through theseal. Thus, once the nozzles 1132 and 1148 are inserted into the noveltyitem 1152 through the seals 1158 and 1160, the contents of the cylinders1128 and 1140 are easily injected. Once the contents are injected,however, the nozzles are removed, and the seals 1158, 1160 return totheir normal biased closed position to prevent the escape of thechemicals from the chambers 1154, 1156.

In yet another alternative embodiment of a cartridge, a dispensingsyringe is shown in FIG. 34 and generally designated 1200. Syringe 1200has a body 1202 which is equipped with a circumferential flange 1204 (ora pair of tabs extending from each side of the body), and a plunger1206. This construction provides for a one-handed operation recharging anovelty item. More specifically, by holding the body adjacent to thecircumferential flange between the index finger and middle finger of auser, and using the thumb to advance the plunger 1206 into the body1202, the entire contents of the dispensing syringe 1200 can be injectedinto the novelty item.

The plunger 1206 has two pistons 1210 and 1208 which are formed withplugs 1212 and 1220 respectively. These plugs 1212 and 1220 are sized tobe snugly received inside the cylinders, e.g., cylinders 1213 and 1221.One cylinder, e.g., cylinder 1213, is filled with dry ingredients 1214and held in place against the seal 1216. Like the cartridges 1000 and1100 discussed above, as piston 1212 is advanced into cylinder 1213, theseal 1216 is ruptured allowing the expulsion of the dry ingredients 1214out of nozzle 1218 and into chamber 1234 of novelty item 1232.

Plug 1220 is positioned in the cylinder 1221 to retain, for example, thefluid 1222 between seal 1224 and plug 1220. As with the cartridgesdiscussed above, as piston 1208 is advanced into the body 1202, fluidpressure is created within the cylinder 1221, thereby rupturing the seal1224. Once the seal is ruptured, the fluid is dispensed, and optionallybathes matrix material 1226 to dissolve the one or more component of thebioluminescence generating system into the fluid. As the piston 1212 isfurther advanced, the fluid 1222 is forced through filter 1228 and outnozzle 1230 and into chamber 1236 of novelty item 1232.

As an alternative to the nozzles 1218 and 1230, a mixing chamber (notshown) can be formed in the body 1202 or attached thereto. Such achamber would provide for the thorough mixing of the dry ingredients1214 and the fluid 1222, prior to introduction of the chemicals into thenovelty item. Such a mixing would be advantageous where it is notfeasible to keep the components of the bioluminescence generating systemseparate until the instant the reaction is desired, such as in asingle-chambered novelty item having a single chemical input port. It isalso to be appreciated that a mixing chamber can be easily formed withinthe cartridge 1000 and/or 1100 or attached thereto.

The charging cartridges 1000, 1100, and 1200 shown and described hereinhave substantially cylindrical chambers within which to store thecomponents of the bioluminescence generating system, separately ortogether, in liquid or solid form. It should be appreciated, however,that any shape chamber is contemplated herein. Specifically, incartridge 1000 and 1100 may be formed with a pair of chambers having arectangular cross-section, or may be formed with each chamber having asemi-circular cross-section, representing one half of a cylindricalblock. Virtually any shape for the block and chambers is contemplatedherein, and the particular embodiments shown in FIGS. 28 through 34 areonly exemplary.

In yet another alternative embodiment (not shown), the cylindricalchambers of the cartridges 1000 and 1100 are replaced by compressibletubing which are positioned within the block and filled with thenecessary chemicals, but are also easily compressed to expel thechemicals within them. The compressible tubing can be made from anyother material which is sufficiently rigid to contain the chemicals,such plastic, rubber or other such material, but pliable enough to allowthe expulsion of the chemicals using a piston. The tubing can be formedin an accordion-shape which has pre-formed creases in the walls of thetubing, or may be formed in any manner which simplifies expulsion of thechemicals. Such a tubing construction would eliminate the need for plugsto retain the chemicals within the block, and will also simplify themanufacturing of the cartridge by eliminating the direct handling of thebioluminescent components.

As an alternative to a cartridge having a block and plunger, a cartridgemay be constructed having a block made from a pliable material thatallows compression of the chemical tubing or other suitable material bysqueezing the sides of the block. In other words, instead of requiring aplunger having pistons which compress the chemical tubing, the block maybe sealed with the chemical tubing contained inside the block, with thechemicals being expelled by squeezing the sides of the block to createthe pressure necessary to rupture the chemical tubing inside.

In addition to a charging cartridge for charging and/or rechargingbioluminescent novelty items, the cartridge incorporating compressibletubing can be formed to allow replacement of the compressible tubingportions within the block. More specifically, once a cartridge has beenused to charge or recharge a novelty item, the compressible tubinghaving a fluid and at least one component of the bioluminescentreaction, and the compressible tubing having the dry ingredients, may beremoved from the block, and a new set of chemical tubing may bepositioned within the block. As a result, the cartridge may berepeatedly used, replacing only the chemical tubing portions. This wouldprovide for the minimization of the costs associated with the use andrepeated use of the novelty items because only the chemical tubingportions would have to be replaced, instead of discarding the entirecartridge following each use.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

Summary of Sequences of Representative luciferases and the reductase setforth in the Sequence Listing

1. SEQ ID NO. 1 Renilla reinformis Luciferase [U.S. Pat. No. 5,418,155]

2. SEQ ID NO. 2 Cypridina hilgendorfii luciferase [EP 0 387 355]

3. SEQ ID NO. 3 Modified Luciola cruciata Luciferase [firefly; U.S. Pat.No. 4,968,613]

4. SEQ ID NO. 4 Vargula (Cypridina) luciferase [Thompson et al. (1989)Proc. Natl. Acad. Sci. U.S.A. 86:6567-6571 and from JP 3-30678 Osaka

5. SEQ ID NO. 5 Apoaequorin-encoding gene [U S. Pat. No. 5,093,240,pAQ440]

6. SEQ ID NO. 6 Recombinant Aequorin AEQ1 [Prasher et al. (1987)"Sequence Comparisons of cDNAs Encoding for Aequorin Isotypes,"Biochemistry 26:1326-1332]

7. SEQ ID NO. 7 Recombinant Aequorin AEQ2 [Prasher et al. (1987)]

8. SEQ ID NO. 8 Recombinant Aequorin AEQ3 [Prasher et al. (1987)]

9. SEQ ID NO. 9 Aequorin photoprotein [Charbonneau et al. (1985) "AminoAcid Sequence of the Calcium-Dependent Photoprotein Aequorin,"Biochemistry 24:6762-6771]

10. SEQ ID NO. 10 Aequorin mutant with increased bioluminescenceactivity [U.S. Pat. No. 5,360,728; Asp 124 changed to Ser]

11. SEQ ID NO. 11 Aequorin mutant with increased bioluminescenceactivity [U.S. Pat. No. 5,360,728; Glu 135 changed to Ser]

12. SEQ ID NO. 12 Aequorin mutant with increased bioluminescenceactivity [U.S. Pat. No. 5,360,728 Gly 129 changed to Ala]

13. SEQ ID NO. 13 Recombinant apoaequorin [sold by Sealite, Sciences,Bogart, Ga. as AQUALITE®, when reconstituted to form aequorin]

14. SEQ ID NO. 14 Vibrio fisheri Flavin reductase [U.S. Pat. No.5,484,723]

What is claimed is:
 1. A combination, comprising:a) an article ofmanufacture; and b) one or more components of a bioluminescencegenerating system and/or a fluorescent protein, whereby the combinationis a novelty item selected from among personal care items, dentifrices,soaps, body paints and powders, and bubble baths.
 2. The combination ofclaim 1, wherein the article of manufacture is selected from among bathpowders, body lotions, gels, body powders, body creams, toothpastes,mouthwashes, soaps, body paints, and cosmetics.
 3. The combination ofclaim 1, wherein the combination comprises a luciferase.
 4. Thecombination of claim 1, wherein the combination comprises a luciferin.5. The combination of claim 1, wherein the combination comprises aluciferin and a luciferase.
 6. The combination of claim 1 that comprisesa fluorescent protein.
 7. The combination of claim 6, wherein thefluorescent protein is a green fluorescent protein, a blue fluorescentprotein or a phycobiliprotein.
 8. The combination of claim 1 that is acosmetic, wherein the cosmetic contains a fluorescent protein.
 9. Thecombination of claim 1 that is for application to the nails, hair, skinor lips.
 10. The combination of claim 1 that is a cosmetic.
 11. Thecombination of claim 10, comprising a delivery vehicle that comprisesleast one component of the bioluminescence generating system.
 12. Thecombination of claim 1 that is nail polish, powder or hair gel.
 13. Thecombination of claim 11, wherein a component is a luciferase.
 14. Thecombination of claim 11, wherein a component is a luciferin.
 15. Thecombination of claim 11, wherein the components comprise a luciferin anda luciferase.
 16. The combination of claim 11, wherein the vehicle is aliposome.
 17. The combination of claim 11, wherein the vehicle is agelatin capsule.
 18. The combination of claim 11, wherein the vehiclecomprises micronized particles of the component(s).
 19. The combinationof claim 11, wherein the vehicle is a time release vehicle.
 20. Thecombination of claim 11, wherein the vehicle is water soluble.
 21. Thecombination of claim 1, wherein the a component of the system isselected from among components of an insect system, a coelenteratesystem, a ctenophore system, a bacterial system, a mollusk system, acrustacea system, a fish system, an annelid system, and an earthwormsystem.
 22. The combination of claim 1, wherein a component of thesystem is selected from among from components of Aequorea, Vargula,Renilla, Obelin, Porichthys, Aristostomias, Odontosyllis, Oplophorus,Gaussia, firefly and bacterial systems.
 23. The combination of claim 1,wherein a component of the system is selected from components of RenillaCavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthussystems.
 24. The combination of claim 1, wherein a component of thesystem is selected Chiroteuthis, Eucleoteuthis, Onychoteuthis,Watasenia; cuttlefish, Sepiolina, Oplophorus, Sergestes, andGnathophausia; Argyropelecus, Yarella, Diaphus, and Neoscopelus systems.25. The combination of claim 1, wherein the bioluminescence generatingsystem comprises a component from a coelenterate.
 26. The combination ofclaim 10, wherein the bioluminescence generating system comprises acomponent from a coelenterate.
 27. The combination of claim 10, whereina component of the bioluminescence generating system is selected fromamong components of Aequorea, Vargula, Renilla, Obelin, Porichthys,Odontosyllis, Aristostomias, Oplophorus, Gaussia, firefly, bacterial,Mnemiopsis, Beroe Gonadostomias, Gaussia, Halisturia, Vampire squid,Glyphus, Mycotophid, Vinciguerria, Howella, Florenciella, Chaudiodus,Melanocostus Sea Pens, mollusc, mushroom, fish, insect, ctenophore andannelid systems.
 28. The combination of claim 10, wherein a component ofthe system is selected from components of Renilla Cavarnularia,Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus systems.
 29. Thecombination of claim 10, wherein the a component of the system isselected from components of Chiroteuthis, Eucleoteuthis, Onychoteuthis,Watasenia; cuttlefish, Sepiolina, Oplophorus, Sergestes, andGnathophausia; Argyropelecus, Yarella, Diaphus, and Neoscopelus systems.30. The combination of claim 10, wherein a component of the system isselected from among components of Aequorea, Vargula, Renilla, Obelin,Porichthys, Aristostomias, Odontosyllis, Oplophorus, Gaussia, fireflyand bacterial systems.